PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 1 / 27
OPEN ACCESS
Ci a ion: Amiama-Roig A, Ba ien os-
Mo eno M, C uz-Zamb ano E, López-Ruiz
LM, González-P ie o R, Rios-O elogio G, e
al. (2025) A R a1-MN–based sys em e eals
new ac o s in ol ed in he escue o b oken
eplica ion o ks. PLoS Gene 21(4): e1011405.
h ps://doi.o g/10.1371/jou nal.pgen.1011405
Edi o : Dmi y A. Go denin, Na ional Ins i u e
o En i onmen al Heal h Sciences, UNITED
STATES OF AMERICA
Recei ed: Augus 26, 2024
Accep ed: Ma ch 10, 2025
Published: Ap il 1, 2025
Pee Re iew His o y: PLOS ecognizes he
bene i s o anspa ency in he pee e iew
p ocess; he e o e, we enable he publica ion
o all o he con en o pee e iew and
au ho esponses alongside inal, published
a icles. The edi o ial his o y o his a icle is
a ailable he e: h ps://doi.o g/10.1371/jou nal.
pgen.1011405
Copy igh : © 2025 Amiama-Roig e al. This is
an open access a icle dis ibu ed unde he
e ms o he C ea i e Commons A ibu ion
License, which pe mi s un es ic ed use,
dis ibu ion, and ep oduc ion in any medium,
RESEARCH ARTICLE
A R a1-MN–based sys em e eals new ac o s
in ol ed in he escue o b oken eplica ion
o ks
Ana Amiama-Roig ☯, Ma a Ba ien os-Mo eno ☯, Es he C uz-Zamb ano,
Luz M. López-Ruiz , Román González-P ie o , Gab iel Ríos-O elogio , Félix P ado *
Cen o Andaluz de Biología Molecula y Medicina Regene a i a (CABIMER), Consejo Supe io de
In es igaciones Cien í icas, Uni e sidad de Se illa, Uni e sidad Pablo de Ola ide, Se ille, Spain
☯ These au ho s con ibu ed equally o his wo k.
* elix.p ado@cabime .es
Abs ac
The in eg i y o he eplica ion o ks is essen ial o an accu a e and imely comple ion o
genome duplica ion. Howe e , li le is known abou how cells deal wi h b oken eplica ion
o ks. We ha e gene a ed in yeas a sys em based on a chime a o he la ges subuni o
he ssDNA binding complex RPA used o he mic ococcal nuclease (R a1-MN) o induce
double-s and b eaks (DSBs) a eplica ion o ks and sea ched o mu an s a ec ed in hei
epai . Ou esul s show ha he co e homologous ecombina ion (HR) p o eins in ol ed
in he o ma ion o he ssDNA/Rad51 ilamen a e essen ial o he epai o DSBs a o ks,
whe eas non-homologous end joining plays no ole. Apa om he endonucleases Mus81
and Yen1, he epai p ocess employs o k-associa ed HR ac o s, b eak-induced eplica-
ion (BIR)-associa ed ac o s and eplisome componen s in ol ed in sis e ch oma id cohe-
sion and o k s abili y, poin ing o eplica ion o k es a by BIR ollowed by o k es o a ion.
No ably, we also ound ac o s con olling he leng h o G1, sugges ing ha a minimal num-
be o ac i e o igins acili a es he epai by con e ging o ks. Ou s udy has also e ealed
a equi emen o checkpoin unc ions, including he syn hesis o Dun1- media ed dNTPs.
Finally, ou sc eening e ealed minimal impac om he loss o ch oma in ac o s, sugges -
ing ha he pa ially disassembled nucleosome s uc u e a he eplica ion o k acili a es
he accessibili y o he epai machine y. In conclusion, his s udy p o ides an o e iew o
he ac o s and mechanisms ha coope a e o epai b oken o ks.
Au ho summa y
The cellula mechanisms ha espond o b oken eplica ion o ks emain poo ly un-
de s ood, despi e he ac ha genomic ins abili y a ising du ing DNA eplica ion is a
hallma k o ea ly cance p og ession. A majo limi a ion in add essing his gap is he ab-
sence o obus sys ems o sys ema ically sc een o he gene ic ac o s in ol ed. Recen ly,
gene ic sys ems ha e been de eloped o induce eplica ion o k b eakage ia a DNA
nick—an in e media e s ep ha is physiologically ele an in DNA epai and opological
egula ion. Howe e , he cellula esponse o di ec double-s and b eaks (DSBs) a
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 2 / 27
PLOS Gene icS B oken o k epai in yeas
eplica ion o ks, such as hose esul ing om o k collapse o unscheduled nuclease
ac i i y, emains la gely unexplo ed. In his s udy, we enginee ed a chime ic p o ein,
R a1-MN, which uses he la ges subuni o he single-s anded DNA-binding complex
RPA wi h mic ococcal nuclease (MN). This chime a p e e en ially gene a es DSBs a
eplica ion o ks, enabling us o sc een o mu an s impai ed in o k epai . Ou sc een-
ing iden i ied no el ac o s ha highligh he signi icance o e o -p one b eak-induced
eplica ion (BIR) es a , o k es o a ion om BIR-in e media es and escue by con e g-
ing o ks. Speci ically, ecombina ion ac o s associa ed wi h eplica ion o ks, eplisome
componen s c i ical o o k s abili y, and egula o s o he G1 phase—con olling epli-
ca ion o igin numbe — a e po en ial playe s o egula e he e iciency o hese pa hways
and he impac o b oken o k epai on genome in eg i y.
In oduc ion
DNA eplica ion duplica es he genome du ing he S phase o he cell cycle. This essen ial
p ocess equi es he coo dina ed i ing o mul iple eplicons wi h bidi ec ional eplisomes
copying la ge genomic egions. The in eg i y o he eplica ion o k is h ea by i s in in-
sically agile molecula na u e (a dynamic nucleosome- ee s uc u e wi h DNA ends and
single-s anded DNA; ssDNA) and he p esence o mul iple ac o s ha hampe i s ad ance
(DNA adduc s, abasic si es, ibonucleoside monophospha es ( NMPs), speci ic DNA s uc-
u es like G-quad uplexes o R-loops, o he p ocesses like ansc ip ion and unbalanced
supplies o deoxynucleoside iphospha es (dNTPs) o his ones) [1]. Dealing wi h hese si u-
a ions is c i ical no only o a imely comple ion o genome duplica ion bu also o p e en
gene ic ins abili y. Acco dingly, cells a e endowed wi h di e en mechanisms ha p o ec and
epai s alled o ks [2–5]. Much less is known, hough, abou he mechanisms ha deal wi h
double-s and b eaks (DSBs) a o ks despi e DSBs a linea molecules a e one o he mos del-
e e ious DNA lesions and hei epai has been ex ensi ely s udied om yeas o human [6–9].
The eason is ha mos o hose s udies ook ad an age o DNA sequence-speci ic endonucle-
ases ha allowed o ollow he epai p ocess; in con as , DSBs a o ks a e sp ead along he
genome a di e en posi ions in each cell, making di icul hei analysis.
DSBs a o ks ha e been p oposed o be epai ed by b eak-induced eplica ion (BIR), a
homologous ecombina ion (HR) p ocess in which he homology is es ic ed o one end;
upon in asion o a homologous empla e, DNA syn hesis can p oceed o la ge genomic
egions [10]. BIR, which has been ex ensi ely cha ac e ized in yeas , does no assemble a
canonical o k; ins ead, i p oceeds h ough a conse a i e DNA syn hesis mechanism ha
is associa ed wi h a mig a ing bubble-like eplica ion o k in which he Polδ subuni Pol32
becomes essen ial [11]. This s uc u e is highly mu agenic and uns able, leading o mul iple
empla e-swi ching e en s and genome ea angemen s ha esemble hose occu ing in
cance genomes [12,13]. In e ms o epai p o eins, he mos ele an di e ence wi h o he
DSB-induced HR e en s is ha i can occu – hough mo e ine icien ly – in he absence
o Rad51 [14]. In acco dance wi h BIR ac ing upon DSBs a o ks, mu an s de ec i e in
eplica ion- coupled nucleosome assembly accumula e b oken o ks ha a e escued by a
Rad52-dependen , Rad51-independen HR mechanism [15]. Howe e , i is unknown i his
equi emen is speci ic o b oken o ks unde condi ions o al e ed ch oma in.
A majo handicap o associa e BIR wi h b oken o ks is ha he sys ems o s udy BIR ollow
he epai o a DNA sequence-speci ic endonuclease-induced DSB wi h a homologous sequence
loca ed on an ec opic egion. As an al e na i e, gene ic sys ems ha e been used in which an
induced nick is con e ed in o a DSB when encoun e ed by a eplica ion o k [16–23]. DNA
p o ided he o iginal au ho and sou ce a e
c edi ed.
Da a a ailabili y s a emen : All ele an da a
a e wi hin he manusc ip and i s Suppo ing
in o ma ion iles.
Funding: This wo k was suppo ed by
Minis e io de Ciencia e Inno ación (MCIN/
AEI/10.13039/501100011033) and
Eu opean Regional De elopmen Fund
(ERDF A way o making Eu ope) (g an
PID2021-127486NB-100 o FP), and
Minis e io de Ciencia e Inno ación (MCIN/
AEI/10.13039/501100011033) and Eu opean
Social Fund (ESF in es ing in you u u e)
(p e-doc o al ellowship PRE2022-101266
o AA-R). The unde s had no ole in s udy
design, da a collec ion and analysis, decision o
publish, o p epa a ion o he manusc ip .
Compe ing in e es s: The au ho s ha e
decla ed ha no compe ing in e es s exis .
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 3 / 27
PLOS Gene icS B oken o k epai in yeas
nicks a e physiologically ele an because hey a e common in e media es o DNA epai and
opological p ocesses ha a e a ge ed in he apeu ic ea men s in cance . A nick a he leading
empla e causes a single-ended DSB (seDSB) ha is escued by an e o -p one BIR-like p ocess
[17–20]; howe e , BIR-associa ed syn hesis is limi ed by wo compensa o y mechanisms:
clea age by he Mus81 endonuclease o con e he D-loop in o a canonical o k and a i al
o a con e ging o k [17]. Ac ually, he a i al o a con e ging o k be o e BIR migh explain
he de ec ion o double-ended DSB (deDSBs) a some nicks a he leading empla e [20–22]. A
nick a he lagging s and also leads o a seDSB using nicked plasmids in Xenopus egg ex ac s
[19], and acco dingly, DNA nicks in bo h leading and lagging s and empla es can igge BIR
[22]. Howe e , nickase-induced nicks a he lagging empla e can be bypassed by he eplisome
in yeas and mammalian cells lea ing a deDSB behind he o k [20–23]. This bypass, hough,
depends on he nickase and s uc u e o he DNA nick [19,20,22], indica ing ha he o k can
espond o he nick and/o he nickase wi hou collapsing.
Replica ion o ks can also collapse and b eak di ec ly unde gene ic o en i onmen al con-
di ions ha cause eplica i e s ess as hose occu ing du ing umou de elopmen [24]. An in
i o app oach o his ype o DNA lesions ea ed Xenopus egg ex ac s wi h ssDNA-speci ic
endonucleases such as S1 o mung bean nuclease, which cu p e e en ially a he o k whe e
ssDNA accumula es unde unpe u bed condi ions. This s udy showed ha he eplisome is
pa ially disman led a e o k b eakage bu ully e-es ablished by a HR p ocess ha equi es
he nuclease ac i i y o M e11, Rad51 and he ini ial DNA syn hesis ac i i y o Polε [25].
In his s udy, we ha e de eloped an in i o sys em ha induces DSBs p e e en ially a
he eplica ion o ks and sea ched o mu an s de ec i e in hei epai . This gene ic analysis
demons a ed ha he HR ac o s in ol ed in he o ma ion o he ssDNA/Rad51 nucleo il-
amen a e essen ial o he epai o DSBs a o ks. In con as o canonical DSBs a linea mol-
ecules, he epai o DSBs a o ks is acili a ed by o k-associa ed HR ac o s, BIR-associa ed
ac o s, eplisome componen s and a imely G1 phase. These esul s sugges ha cells deal
wi h seDSBs a b oken o ks by wo mechanisms: BIR ollowed by o k es o a ion and escue
by con e ging eplica ion o ks, and e eal new playe con olling hei e iciency.
Resul s
The chime a R a1-MN p o ides a gene ic sys em o s udy he epai o
DSBs a eplica ion o ks
Ch oma in endogenous clea age (ChEC) p o ides a me hod o de ec p o ein ch oma in
binding [26]. This assay is based in he exp ession o a chime a o he p o ein o in e es wi h
he mic ococcal nuclease (MN), whose nucleoly ic ac i i y is ac i a ed wi h Ca2+ ions. I he
p o ein is bound o DNA, ac i a ion o he MN domain will induce a de ec able cu (Fig 1A,
le ). Since he in acellula le els o Ca2+ ions a e low o MN ac i a ion, his assay equi es
cells o be pe meabilized wi h digi onin ollowed by addi ion o CaCl2 (S1A Fig). This assay
has also been used o ollow he binding o epai p o eins o non-DSBs DNA lesions, as he
ssDNA agmen s gene a ed by he encoun e o eplica ion o ks wi h me hyl me hanesul on-
a e (MMS)-induced DNA adduc s [27–33].
An example o his app oach is he chime a R a1-MN, which con ains he la ges subuni
o he ssDNA binding complex RPA. RPA is an essen ial complex in ol ed in eplica ion
o k s abili y, DNA epai and checkpoin ac i a ion [34,35]. A e ea ing pe meabilized
cells wi h CaCl2 o di e en imes, o al DNA was ex ac ed and un in o an aga ose gel. As
expec ed, he ex en o DNA diges ion was exace ba ed in cells ea ed wi h 0.005% MMS
(S1A Fig); howe e , in con as o o he chime as like Rad52-MN ha equi es 30 minu es in
he absence o MMS [27], DNA diges ion by R a1-MN was de ec ed a e 5 minu es o CaCl2
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 4 / 27
PLOS Gene icS B oken o k epai in yeas
Fig 1. The chime a R a1-MN p o ides a gene ic sys em o s udy he epai o DSBs a eplica ion o ks. (A) ChEC analysis o RFA1-MN cells a es ed in G1 wi h
α- ac o and eleased in o S phase o 30 minu es. To al DNA om cells pe meabilized and ea ed wi h 2 mM CaCl2 o di e en imes is shown, as well as he FACS
p o iles. A scheme wi h he a ional o he ChEC app oach is shown on he le . (B) 2D/ChEC analysis o eplica ion in e media es o RFA1-MN cells synch onised in
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 5 / 27
PLOS Gene icS B oken o k epai in yeas
ea men . This diges ion was obse ed bo h in cells main ained in G1 wi h α-
ac o and eleased in o S phase o 30 minu es, al hough he kine ics o DNA diges ion was
as e in S phase cells (Fig 1A, compa e 10 minu es diges ion). This esul is consis en wi h
RPA localiza ion a he ansc ibed egions o ac i e genes [36]. Howe e , RPA accumula es
p e e en ially a eplica ion o ks whe e i p o ec s ssDNA unde unpe u bed and s essed
condi ions, as de e mined by ChIP-seq o whole ch omosomes and mic oscopy analyses o
RPA oci in G1 and S phases [37–39]. Since ChEC p e e en ially de ec s DNA b eaks a lineal
molecules as he numbe o o ks ela i e o he whole genome is low, we s udied R a1-MN
binding o eplica ion o ks by 2D/ChEC. In his assay, eplica ion in e media es om ChEC-
ea ed cells a e analysed by 2-dimensional (2D) elec opho esis [28]. Ac i a ion o he MN
ac i i y o R a1-MN wi h Ca2+ diges ed all eplica ion in e media es in less han a minu e (Fig
1B), in sha p con as wi h o he chime as like Rad52-MN o Rad27-MN ha equi ed se e al
minu es o a pa ial diges ion [27]. Thus, al hough R a1-MN can induce DSBs a linea mole-
cules, i p e e en ially diges s eplica ion o ks.
The R a1-MN chime a is exp essed a he same s eady s a e le el as he non- agged p o ein
(Fig 1C) and is p o icien in DNA damage ole ance (Fig 1D), DSB epai ( o al and media ed
by HR) (Figs 1E and S1B), checkpoin ac i a ion (Fig 1F) and DNA eplica ion (Fig 1G). Only
a sligh delay om G1 o G2/M was obse ed by FACS, al hough he budding index and dou-
bling ime we e simila in RFA1-MN and wild- ype cells (S1C Fig). Impo an ly, he ac ha
he RFA1-MN mu an beha es as he wild- ype s ain in he p esence o high concen a ions
o MMS and hyd oxyu ea (HU) indica es ha he chime a is also p o icien in eplica ion o k
p ocessi i y and s abili y e en unde high eplica ion s ess condi ions.
Rema kably, he gene ic combina ion RFA1-MN ad52∆ is le hal as de e mined by gene ic
analyses (Figs 1H and S1D). This syn he ic le hali y sugges s ha R a1-MN causes ecom-
binogenic lesions ha need o be epai ed. HR deals wi h wo di e en DNA lesions: DSB and
eplica ion associa ed-ssDNA. A majo di e ence be ween hem is ha he o me , bu no he
la e , can be epai ed in G2::cRAD52 cells ha es ic he exp ession o Rad52 o G2/M [27].
We obse ed ha he RFA1-MN G2::cRAD52 s ain g ew as he wild ype (Fig 1I), sugges ing
ha R a1-MN causes DSBs. Rad52 essen iali y in R a1-MN-exp essing cells con as s wi h he
non-essen ial ole o HR in he epai o mechanically- and HO endonuclease-induced DSBs
whe e NHEJ can ope a e as a backup mechanism (Fig 1J) [40].
The simples explana ion o hese esul s is ha he amoun o in acellula Ca2+ is su i-
cien o induce he nucleoly ic ac i i y o R a1-MN a a a e ha has no e ec on cell g ow h
unless HR is absen . Al hough we canno disca d he o ma ion o some DSBs a o he
egions, he p e e en ial accumula ion o RPA a eplica ion o ks [37–39], he high e iciency
G1 wi h α- ac o and eleased in o S phase o 30 minu es. To al DNA om cells pe meabilized and ea ed wi h Ca2+ o di e en imes was diges ed wi h speci ic
es ic ion enzymes and analysed by 2D elec opho esis. A schema ic ep esen a ion o he mig a ion pa e n o eplica ion in e media es is shown on he igh .
(C) R a1 exp ession in wild- ype and RFA1-MN cells om exponen ially g owing cul u es as de e mined by wes e n blo analysis. (D) MMS and HU sensi i i y o
RFA1-MN cells. Wild- ype and ad52∆ cells we e included as con ols. (E) DSB sensi i i y o RFA1-MN cells ans o med wi h pGAL-HO and g own in glucose
(GAL1p ep ession) and galac ose-con aining medium (GAL1p ac i a ion). An HO-induced DSB a he MAT locus can be epai ed by NHEJ o , p e e en ially, by HR
wi h he HMR o HML dono . The analysis was pe o med in wild- ype and ku70∆ backg ound (de ec i e in NHEJ). (F) Rad53 ac i a ion in wild- ype and RFA1-MN
cells as de e mined by wes e n blo analysis o exponen ially g owing cul u es ei he in he absence o p esence o 0.005% MMS o 1 hou . (G) Cell cycle p og ession
o wild- ype and RFA1-MN cells synch onised in G1 wi h α- ac o and eleased in o S phase o di e en imes as de e mined by FACS analysis. (H) RFA1-MN ad52∆
le hali y as de e mined by e ad analysis. (I) E ec o es ic ing Rad52 exp ession o G2/M in wild ype (G2::cRAD52) and RFA1-MN cells (RFA1-MN G2::cRAD52).
(J) HO-induced DSB epai in cells de ec i e in HR ( ad52∆) and/o NHEJ (ku70∆). Cells we e ans o med wi h pGAL-HO and g own in glucose (GAL1p ep ession)
and galac ose-con aining medium (GAL1p ac i a ion). (K) P oposed model o he essen ial ole o HR in R a1-MN exp essing cells. (L) E ec o he pi 1∆, pol32∆,
yen1∆, mus81∆ and mus81∆ yen1∆ mu a ions in he g ow h o RFA1-MN cells in he absence and p esence o di e en CaCl2 concen a ions. A high concen a ion,
CaCl2 can o m c ys als ha did no a ec he ep oducibili y o he assay. (D-E, I-J, L) Cell g ow h was de e mined by spo ing 10- old se ial dilu ions o he same
numbe o mid-log g owing cells on o he indica ed mediums. All he analyses we e epea ed a leas wice wi h simila esul s.
h ps://doi.o g/10.1371/jou nal.pgen.1011405.g001
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 6 / 27
PLOS Gene icS B oken o k epai in yeas
o R a1-MN o diges eplica ion o ks, and he essen ial ole o HR o RFA1-MN cell iabili y
sugges ha mos o hese DSBs s em om R a1-MN–cu eplica ion o ks, p e e en ially a
he lagging s and ha accumula es mos ssDNA/RPA (Fig 1K). We do no conside D-loops
as a p e e en ial a ge o R a1-MN-induced clea age, because Rad52 does no p omo e, bu
ins ead p e en s RFA1-MN cells le hali y. Acco dingly, RFA1-MN cells displayed a wild- ype
g ow h in he p esence o high concen a ions o HU e en in pla es en iched wi h CaCl2 o
inc ease he a e o clea age (S1E Fig).
I mos DSBs s em om diges ed eplica ion o ks, he exp ession o R a1-MN should dis-
play syn he ic g ow h de ec s wi h mu a ions p e iously iden i ied as equi ed o he escue o
b oken eplica ion o ks. We es ed he absence o Rad51, M e11, Pol32, Mus81 and Pi 1 (Fig
1L). Jus a ew double mu an s RFA1-MN ad51∆ and RFA1-MN m e11∆ ge mina ed leading
o mic ocolonies ha g ew be e a e s eaking in a new pla e, likely by adap a ion o g ow
wi h lowe le el o in acellula calcium o he selec ion o supp esso s (Figs 2A and S2A). The
g ow h o he RFA1-MN s ain was a ec ed o di e en ex en s in he absence o Pi 1, Pol32
and Mus81, bu only in pla es en iched wi h CaCl2 (Fig 1L). The sensi i i y o mus81∆ o
nick-induced o k b eakage is se e ely agg a a ed in he absence o Yen1 [17,21], an endonu-
clease ha pa icipa es wi h Mus81 in he esolu ion o single Holiday junc ions (HJs) [41]
as hose expec ed om he me ging o a BIR-associa ed mig a ing D-loop wi h a con e ging
o k. The absence o Mus81 and Yen1 s ongly impai ed cell g ow h in R a1-MN-exp essing
cells, al hough wi hou being le hal (Fig 1L). In conclusion, R a1-MN p o ides a gene ic sys-
em o sea ch o ac o s in ol ed in he epai o DSBs a eplica ion o ks.
Iden i ying unc ions equi ed o he epai o DSBs a eplica ion o ks
To sea ch o ac o s equi ed o he epai o DSBs a o ks, we ollowed a Syn he ic Gene ic
A ay (SGA) analysis based in he c ossing o an o de ed a ay o null mu an s o a s ain
ha bou ing he que y allele RFA1-MN and speci ic ma ke s such ha he meio ic p ogeny
wi h bo h he RFA1-MN allele and he null mu a ion can be sco ed o i ness [42]. This cus-
omized a ay encompasses 358 null mu an s selec ed acco ding o hei con i med o pu a i e
connec ion wi h he DNA damage esponse (S1 Table). The loss o iabili y o cell i ness was
sco ed in pla es wi hou and wi h 400 mM CaCl2 o inc ease he sensi i i y o he sc eening.
We ob ained 62 hi s, ou o which 6 we e wild ype o he expec ed null mu a ion and 4 could
no be alida ed by PCR (S2 Table). Fo manual inspec ion o hese gene ic in e ac ions,
we c ossed he o iginal RFA1-MN s ain wi h each null mu an (including hose sco ed as
syn he ically le hal), analysed gene ically he dissec ed spo es and s udied he loss o i ness
by d op assays in medium wi hou and wi h di e en concen a ions o CaCl2. This s udy
e ealed 44 genes ha a e equi ed o a g ea e o lesse ex en o he iabili y o R a1-MN
exp essing cells (S2 Table). Excep o ad52∆ and pm 1∆ mu an s, we ob ained double
mu an s wi h he RFA1-MN allele o he es , including hose sco ed as syn he ically le hal in
he SGA sc eening. These “le hal” mu an s included ad51∆ and m e11∆ and displayed a sim-
ila beha iou (Figs 2A and S2A). Pm 1 is a Ca2+/Mn2+ ATPase equi ed o Ca2+ anspo
o Golgi whose null mu an accumula es excess Ca2+ ions [43], which is likely causing a le hal
numbe o b oken o ks.
The epai o DSBs a eplica ion o ks equi es DSB- and eplica ion o k-
speci ic HR ac i i ies
Apa om he media o Rad52, we sco ed as syn he ically le hal he MRX complex (M e11,
Rad50 and X s2), he ecombinase Rad51 and i s helpe s Rad55 and Rad54 (Fig 2A and S2
Table), which a e essen ial componen s o he HR machine y dealing wi h DSBs [44]. The
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 7 / 27
PLOS Gene icS B oken o k epai in yeas
Fig 2. The epai o DSBs a eplica ion o ks equi es DSB- and eplica ion o k-speci ic HR ac i i ies. (A) Syn he ic le hali y o
RFA1-MN wi h he indica ed mu an s as de e mined by e ad analysis. (B–F) E ec o he indica ed mu a ions in he g ow h o RFA1-MN
cells as de e mined by spo ing 10- old se ial dilu ions o he same numbe o mid-log g owing cells on o SMM medium wi hou o wi h he
indica ed concen a ions o CaCl2. The analyses we e epea ed a leas wice wi h simila esul s. Mu an s sco ed in he SGA sc eening a e
shown in bold.
h ps://doi.o g/10.1371/jou nal.pgen.1011405.g002
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 8 / 27
PLOS Gene icS B oken o k epai in yeas
ecombina ion p o ein Rad59 was also ound in he sc eening bu only in he p esence o
CaCl2 (Fig 2B), which is consis en wi h he mino e ec o ad59∆ in HR in he p esence o
Rad51 [45].
Ano he hi sco ed as syn he ically le hal was R 105 (Fig 2A and S2 Table), despi e i
encodes a chape one in ol ed in he ans e o he nucleus and deposi ion a ssDNA o he
RPA complex. Howe e , he educ ion in he le el o RPA a o ks in 105∆ cells is sligh
unde no mal condi ions [37,46], which migh explain why R a1-MN is inducing DNA
damage as in e ed om he le hali y. The syn he ic le hali y o RFA1-MN 105∆ cells migh
be ela ed o he unc ion o R 105 in HR – whe e i acili a es Rad51 loading a ssDNA o
DSB-induced gene con e sion and BIR [46] – and o a lesse ex en o he ole o RPA in
eplica ion o k s abili y and checkpoin ac i a ion (see below). Ac ually, he DSB epai de ec
in 105∆ is almos as s ong as ha displayed by ad52∆ (S2B Fig). Rema kably, ssDNA s a-
biliza ion by RPA is pa icula ly c i ical o HR mechanisms ha in ol es long-li ed ssDNA
in e media es, in pa icula BIR [47]. Al oge he , hese esul s demons a e ha he MRX
complex, he Rad51/ssDNA nucleo ilamen and he ac o s ha p omo e i s assembly a e
essen ial o he epai o DSBs a o ks.
Du ing DSB-induced HR, Sae2 p omo es he nuclease ac i i y o he MRX complex in
he ini ial p ocessing o DSB ends o gene a e sho s e ches o 3’-ended ssDNA. This DNA
esec ion is comple ed by he nuclease and helicase ac i i ies o Exo1 and Sgs1/Dna2 h ough
complemen a y mechanisms [48]. In con as o MRX, Sae2 was iden i ied in he sc eening
only in he p esence o CaCl2 (Fig 2B). To add ess he ele ance o long esec ion, we analysed
he e ec on cell g ow h o he single and double exo1∆ and sgs1∆ mu an s in combina ion
wi h RFA1-MN. Only he RFA1-MN exo1∆ sgs1∆ displayed a loss o g ow h in he p esence
o CaCl2 (Fig 2B). Again, he lack o long esec ion was no essen ial. I is wo h no ing he
lack o e ec o sgs1∆, because he helicase Sgs1 is equi ed o he dissolu ion o double HJ
(dHJ) and sis e -ch oma in junc ion (SCJ) s uc u es by he Top3/Sgs1/Rmi1 complex [49,50].
Rema kably, ano he hi o he sc eening was he opoisome ase Top3, sco ed as le hal (S2
Table). Since we could no alida e he collec ion mu an by PCR, we gene a ed he RFA1-MN
op3∆ mu an by gene ic c oss. The lack o Top3 caused a g ow h de ec in he p esence o
CaCl2 (Fig 2B), sugges ing a Sgs1-independen ole in he escue o b oken o ks.
Two componen s o he helpe Shu complex (Psy3 and Csm2) we e sco ed as syn he ically
sick in he p esence o CaCl2 (Fig 2C). The Shu complex is also in ol ed in Rad51 ilamen
o ma ion [51], bu in con as o he a o emen ioned HR mu an s, shu mu an s a e p ima ily
sensi i e o MMS-induced eplica ion-associa ed ssDNA lesions bu no o DSB-inducing
agen s [52,53].
Ano he unc ional gene ic hub iden i ied in he SGA sc eening is o med by 109∆,
107∆, mms1∆ and mms22∆ (Fig 2D) [54,55]. R 109 is a his one ace yl ans e ase ha
ace yla es his one H3 a lysine 56 (H3K56) [56,57], which in u n acili a es his one H3/H4
deposi ion by inc easing i s in e ac ion wi h ch oma in assembly ac o s CAF and R 106
[58]. This pa hway is s imula ed a e ubiqui yla ion o he ace yla ed his one by he R -
101Mms22/Mms1 complex [59], which is associa ed wi h he eplisome du ing S phase [60]. The
in ol emen o he ubiqui in ligase R 101 was con i med by manual inspec ion o RFA1-MN
101∆ mu an s (Fig 2D). A ch oma in, he H3K56ac/ R 101Mms22/Mms1 pa hway p omo es
he ecombina ional epai o eplica ion-associa ed ssDNA lesions bu no o DSBs [61–64].
The ch oma in assembly and ecombina ional unc ions o H3K56ac can be sepa a ed in a
double mu an cac1∆ 106∆ (Cac1 encodes he la ges subuni o he CAF complex) because
he abili y o H3K56ac o s imula e nucleosome assembly depends on CAF and R 106 [58],
whe eas i s abili y o p omo e HR is independen o CAF and R 106 [65–67]. The iple
RFA1-MN cac1∆ 106∆ was ha dly a ec ed e en a high concen a ions o CaCl2 (S2C Fig),
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 9 / 27
PLOS Gene icS B oken o k epai in yeas
sugges ing ha ch oma in assembly does no play a majo ole in he epai o DSBs a o ks.
This was u he con i med by es ing he sp 16-m allele (alone o in combina ion wi h cac1∆
106∆), which impai s he eplica ion-coupled nucleosome ac i i y o he FACT complex
[68], and he pol1-2A2, mcm2-3A and dpb3∆ alleles, de ec i e in he ans e o pa en al
his ones o nascen s ands [69–71]. Only he RFA1-MN pol1-2A2 mu an displayed a weak
e ec in pla es wi h high CaCl2 concen a ions (S2C Fig), which migh be ela ed o a sub le
de ec a i s polyme ase ac i i y. The ecombina ional ole o H3K56ac in he epai o DSBs
a o ks was u he suppo ed by he inding o he hs 3∆ mu an in he sc eening (Fig 2D).
Hs 3 o ms wi h Hs 4 a Si uin complex ha deace yla es ch oma in-deposi ed H3K56ac once
he eplica i e DNA damage is epai ed [72,73]. Al e na i ely, he g ow h de ec o RFA1-MN
hs 3∆ cells migh be due o he inhibi o y e ec o H3K56 hype -ace yla ion on DNA syn he-
sis du ing BIR [74].
R 109 also acili a es he ec ui men o s alled o ks o R 107 h ough a H3K56ac-
independen mechanism [75]. R 107 is a p o ein ha ac s as a sca old o h ee genome
main enance complexes: he R 101Mms22/Mms1 ubiqui in ligase, he Slx4 sca old o he Slx1
and Mus81-Mms4 nucleases and he Smc5/6 SUMO ligase [76,77]. To add ess he ole o he
Smc5/6 SUMO ligase we es ed he smc6–56 allele and obse ed no e ec on he g ow h o
R a1-MN exp essing cells (S2D Fig). This esul is consis en wi h he dispensabili y o Sgs1
in b oken o k epai , as he Smc5/6 complex is also equi ed o MMS-induced SCJ dissolu-
ion and DSB epai [78]. We ha e u he disca ded a ole o hese s uc u es in he epai
o DSBs a o ks by analysing he e ec o a ad18∆ mu an , de ec i e in PCNA ubiqui yla-
ion and eplica ion s ess-associa ed SCJ o ma ion (S2D Fig) [79,80]. The e o e, he ole o
R 109 and R 107 on he g ow h o R a1-MN exp essing cells is associa ed wi h he ubiqui yl-
a ion and nuclease unc ions o R 101Mms22/Mms1 and Mus81, espec i ely. In line wi h he la e
unc ion, i is pa icula ly in e es ing he inding o Rad27 in he SGA sc eening (Fig 2E), as
he physical and unc ional in e ac ions o his endonuclease wi h Slx4-Mus81 (including syn-
he ic le hali y o he double null mu an s) migh be c i ical o he esolu ion o in e media es
du ing eplica ion s ess [81]. Al oge he , hese esul s indica e ha he epai o DSBs a o ks
equi es eplica ion o k-speci ic HR ac i i ies.
These esul s demons a e ha DSB- and eplica ion- o k-associa ed HR unc ions pa ici-
pa e in he epai o b oken eplica ion o ks. On he con a y, NHEJ seems no o be equi ed
o b oken o k epai because nei he he Ku70/Ku/80 complex no Nej1 we e sco ed as posi-
i e hi s. This was con i med by manual inspec ion o a RFA1-MN ku70∆ mu an (Fig 2F).
Checkpoin ac o s acili a e he epai o DSBs a o ks
The second unc ional g oup in ol ed in he epai o DSBs a o ks encompasses se e al
DNA damage checkpoin (DDC) (Rad9, he 9-1-1 complex (Ddc1/Mec3/Rad17) and i s
loade Rad24) (Fig 3A) and DNA eplica ion checkpoin (DRC) ac o s (M c1, To 1, and he
C 8 and Dcc1 componen s o he PCNA loade RFC/C 18/C 8/Dcc1 (C 18-RFC complex))
(Fig 3B) [82,83]. All hese ac o s ha e addi ional unc ions apa om checkpoin ac i a ion:
Rad9 p o ec s DSBs om p ema u e esec ion [84]; he 9-1-1 complex pa icipa es in DDT
and eplica ion-coupled nucleosome assembly [85,86]; M c1 and To 1 ha e oles in coupling
helicase and polyme ase ac i i ies, sis e ch oma id cohesion (SCC), and in he case o To 1,
s able o k pausing a eplica ion o k blocks [87–91]; he C 18-RFC complex is equi ed o
eplica ion o k s abili y upon s ess and SCC [92,93]. In his case, since he anno a ed c 18∆
mu an was wild ype in he collec ion, we gene a ed a new one and obse ed ha i did no
a ec R a1-MN g ow h e en a high CaCl2 concen a ions (Fig 3B). This esul is consis en
wi h p e ious obse a ions showing ha C 18, C 8 and Dcc1 a e equi ed o MMS and
HU esis ance, bu only C 8 and Dcc1 a e equi ed o ionizing adia ion (IR) and UV ligh
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 16 / 27
PLOS Gene icS B oken o k epai in yeas
p ocessing o he D-loop [81]. I is impo an o ema k ha he g ow h de ec s obse ed in
he absence o eplica ion ac o s a e unlikely due o an accumula ion o ssDNA and a highe
p obabili y o o k b eakage by R a1-MN o o he addi i e e ec s o o k clea age and eplica-
ion s ess because he RFA1-MN mu an beha es bo h wi h and wi hou calcium as he wild-
ype s ain e en in he p esence o high concen a ions o MMS and HU ha s ongly impai
cell g ow h (Figs 1D and S1E).
The es ablishmen o cohesion is achie ed h ough wo pa ially complemen a y mecha-
nisms: he con e sion o cohesins associa ed wi h un eplica ed DNA ahead o he o k in o
cohesi e s uc u es behind he o k (dependen on C 4, To 1/Csm3 and Chl1) and he loading
o nucleoplasmic cohesins on o o k-associa ed nascen DNA (dependen on he cohesin
loade Scc2/Scc4 and he C 8-RFC complex) [90]. Ou esul s show ha he absence o Chl1
o C 18 does no impac he epai o DSBs a o ks. Thus, he ole o C 4, M c1, C 8, Dcc1
and he To 1/Csm3 complex in b oken o k epai canno be explained jus by a de ec in
SCC. Conse a i e eplica ion associa ed wi h D-loop mig a ion uncouples he leading and
lagging s ands [118]. In a canonical o k, hey a e coupled h ough physical in e ac ions o
C 4, M c1 and To 1/Csm3 wi h he CMG helicase and he DNA polyme ases Pol ε and Pol
α [87,119]. A po en ial ea angemen o hese in e ac ions in he mig a ing D-loop s uc u e
migh be ela ed o he ecombina ional ole o he R 109/H3K56ac/ R 101Mms22/Mms1 pa h-
way, as he sensi i i y o eplica ion s ess o cells lacking his pa hway can be supp essed by
mu a ions in C 4, M c1, Dpb4 (Pol ε) o Mcm6 ha uncouple he CMG helicase om he
DNA polyme ases [60,66]. A sc eening o ac o s in ol ed in he escue o oncogene-induced
s essed o ks unco e ed, oge he wi h he BIR p o eins Rad52 and PolD3 (human o holog
o Pol32), he FPC componen s Tipin and Timeless (human o hologs o To 1 and Csm3)
[120], sugges ing a conse a ion o hese ac o s.
Fou h, sho ening o he G1 phase comp omises he escue o b oken o ks, as in e ed by
he in e ed co ela ion be ween G1 leng h and cell g ow h de ec s in RFA1-MN cells lacking
di e en inhibi o s o he G1/S ansi ion. Con e ging o ks limi he mu agenici y associa ed
wi h he epai o a nick-induced DSB, likely by me ging wi h he D-loop [17]. Since a p ema-
u e en y in o S phase educes he numbe o licensed o igins [104,105], he se e e g ow h
de ec s o R a1-MN-exp essing cells in combina ion wi h a sho ening o G1 migh be due o a
educ ion in he numbe o ac i e o ks ha could escue he b oken o ks. In yeas and cance
cells, p ema u e en y in o S phase by CDK de egula ion in G1 causes a educ ion in he num-
be o ac i e eplica ion o igins and genome ins abili y. This ins abili y has been p oposed o
esul om a highe equency o o k collapse and/o he en y in o mi osis wi h incomple ely
eplica ed genomes [121]. Ou esul sugges s ha i may also a ise om un epai ed b oken
o ks and/o excess BIR-induced mu agenesis.
Fi h, he epai o DSBs a o ks by HR is an e icien p ocess. The le hali y o he double
mu an RFA1-MN ad52∆ sugges s ha a leas one o k pe cell cycle is cu by he chime a.
Howe e , RFA1-MN cells did no display g ow h de ec s and he checkpoin was no equi ed
excep a high le els o CaCl2 (consis en wi h an accumula ion o DSBs and/o BIR- associa ed
ssDNA) o in he absence o Rad52 (consis en wi h he accumula ion o DNA esec ion-
media ed ssDNA a b oken o ks when s and exchange is abolished [107]). In e es ingly,
we ha e ound Dun1 in ou sc eening and demons a ed ha he g ow h de ec is due o a
educ ion in he le els o dNTPs, in line wi h he Dun1-dependen inc ease in bo h dNTPs
and mu agenesis obse ed du ing BIR [12].
Taking in o accoun ou esul s and p e ious s udies, we p opose he ollowing model o
he epai o seDSBs a o ks (Fig 5). A Rad51/ssDNA nucleo ilamen o med a he b oken
nascen s and would in ade he sis e ch oma id in he con ex o he eplisome machin-
e y wi h he help o s alled o k-associa ed HR ac o s, leading o he o ma ion o a D-loop
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 17 / 27
PLOS Gene icS B oken o k epai in yeas
s uc u e. This in asion s ep has o occu behind he CMG helicase, which may be e ained a
he p oximi y oge he wi h eplisome componen s o u he es o a ion o he eplica ion
o k. These eplisome componen s migh be equi ed o he s abili y o he mig a ing D-loop
(whose ad ance would equi e Pol32 and Pi 1) and/o he con e sion o his s uc u e in o a
canonical o k upon he ac i i y o Mus81. Cohesins would also con ibu e o he s abili y o
his s uc u e and/o o he p e ious in asion s ep. Replica ion o k es a by his BIR-like
mechanism is associa ed wi h high le els o mu agenesis and empla e swi ching e en s. This
gene ic ins abili y would be po en ially es ic ed by speci ic ac o s like Rad27 and Top3,
he con e sion o he D-loop in o a canonical o k and he me ging wi h a con e ging o k,
a ou ed by he licensing o su icien eplica ion o igins du ing G1 phase. In his con ex ,
he nucleases Mus81 and Yen1 migh also be equi ed o he esolu ion o he HJ s uc u e
gene a ed a e o k me ging. A majo obse a ion o his s udy is he essen ial ole o he HR
machine y. We hink ha HR-media ed s and exchange would no only p omo e eplica ion
o k es a , bu would also p e en inhibi ion o o igin i ing by checkpoin ac i a ion, as
eplica ion is equi ed o he escue by con e ging o ks.
Apa om he posi i e hi s, some o which equi es u he in es iga ion o unde s and
hei connec ion wi h b oken o k epai (S4C Fig), ou sc eening e ealed a sca ce impac by
he loss o ch oma in ac o s. This is unexpec ed aking in o accoun hei ele ance du ing
DNA eplica ion and DSB epai [122]. Mu an s a ec ing he deposi ion o newly and pa en al
his ones du ing eplica ion ha dly a ec ed he iabili y o R a1-MN-exp essing cells. Likewise,
his one chape ones ha pa icipa e in eplica ion-independen nucleosome exchange (HIR,
Nap1, Chz1) and ch oma in emodelling ac o s (INO80, SWR1, ISW1, ISW2, SWI/SNF and
RSC) we e nega i e hi s in he sc eening, wi h he excep ion o Chd1 (S2 Table and S4D Fig).
Al hough he in ol emen o ch oma in in he epai o DSBs a o ks equi es a mo e de ailed
analysis, one possibili y o explain i s low impac is ha he pa ially disassembled nucleosome
s uc u e a he ad ancing o k acili a es he accessibili y o he epai machine y.
A limi a ion o ou sys em is ha many o he hi s we e iden i ied by adding CaCl2 o
he medium o inc ease he numbe o b oken o ks. This sudden inc ease in cy osolic Ca2+
igge s he ep og amming o Ca2+ anspo e s o es o e physiological le els [123,124].
Thus, we canno ule ou ha some o he hi s migh be speci ic o his Ca2+ s ess con ex .
Mo eo e , he clea age likely occu s p e e en ially a he lagging s and, whe e RPA ends o
accumula e. I will be in e es ing o de e mine he e ec o he analysed mu an s i he DSB
occu s p e e en ially a he leading s and.
In summa y, ou esul s p o ide new gene ic equi emen s o he epai o b oken
o ks and highligh he signi icance o e o -p one BIR es a , o k es o a ion om BIR-
in e media es and escue by con e ging o ks. Speci ically, ecombina ion ac o s associa ed
wi h eplica ion o ks, eplisome componen s c i ical o o k s abili y, and egula o s o he
G1 phase may po en ially con ol he e iciency o hese pa hways and he impac o b oken
o k epai on genome in eg i y, especially in egions wi h low densi y o ac i e o igins like
he end o ch omosomes and common agile si es (CFS) in mammalian genomes [125,126],
which elies on BIR-like mechanism: MiDAS (mi o ic DNA syn hesis) and ALT (al e na i e
leng hening o elome es) [127]. Fu u e molecula expe imen s will be equi ed o es he
di e en scena ios in e ed om ou gene ic analyses.
Ma e ials and me hods
Yeas s ains, plasmids and g ow h condi ions
All Saccha omyces ce e isiae s ains used a e haploid de i ed om BY4741 o W303. Yeas
s ains used in his s udy a e lis ed in S3 Table. Mos s ains we e gene a ed by gene ic c osses.
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 18 / 27
PLOS Gene icS B oken o k epai in yeas
Tagged and dele ion s ains we e cons uc ed by a PCR-based s a egy [128]. pDML5 is a
URA3-based cen ome ic plasmid ha exp esses RAD52 om he galac ose-inducible GAL1
p omo e . pGAL-HO is a URA3-based mul icopy plasmid exp essing he endonuclease HO
om he GAL1 p omo e [129]. Yeas cells we e g own in supplemen ed minimal medium
(SMM) a 30 °C excep o liquid cul u es supplemen ed wi h 400 mM CaCl2, which was
pe o med a 26 °C o educe Ca2+ p ecipi a ion. Fo G1 synch oniza ion, cells we e g own o
mid-log phase and α- ac o was added wice a 60 min in e als a ei he 1 (BAR1 s ains) o
0.5 μg/ml (ba 1∆ s ains). Then, cells we e washed h ee imes and eleased in o esh medium
wi h 50 μg/ml p onase.
Syn he ic gene ic a ay analysis
The syn he ic gene ic a ay analysis (SGA) was pe o med as epo ed wi h some modi ica ions
[42]. The que y s ains (RFA1-MN::NAT and con ol p1∆::NAT) we e c ossed wi h a cus omized
a ay o null mu an s using a manual eplica o . The double mu an s wi h RFA1-MN we e sco ed
as syn he ically le hal o syn he ically sick by compa ing hei g ow h wi h he double mu an s
wi h p1∆::NAT on he SDMSG-His/A g/Lys-cana anine- hialysine-G418-nou seo h icin pla es.
To add ess he e ec o Ca2+, bo h se s o double mu an s we e i s eplica pla ed o SMM and
hen o SMM supplemen ed wi h 400 mM CaCl2.
DNA damage sensi i i y
The sensi i i y o R a1-MN exp ession, zeocin, MMS, HU and HO exp ession was de e -
mined by spo ing en- old se ial dilu ions o he same numbe o mid-log g owing cells on o
SMM medium wi hou o wi h CaCl2, zeocin, MMS and HU, o on o glucose and galac ose-
con aining medium (HO). Fo ionizing adia ion sensi i i y spo ed cells we e i adia ed
and hen g own unde unpe u bed condi ions. All analyses we e epea ed a leas wice wi h
simila esul s.
Cell g ow h analyses
Cell cycle was ollowed by DNA con en . DNA con en analysis was pe o med by low
cy ome y as epo ed p e iously [130]. Cells we e ixed wi h 70% e hanol, washed wi h
phospha e-bu e ed saline (PBS), incuba ed wi h 1 mg o RNaseA/ ml PBS, and s ained wi h
5 μg/ml p opidium iodide. Samples we e sonica ed o sepa a e single cells and analyzed in a
FACSCalibu low cy ome e . The budding index (pe cen age o cells wi h bud) was de e -
mined by coun ing 100 cells a each ime poin and eplica e. The doubling ime was calcu-
la ed by measu ing he OD600 om exponen ially g owing cul u es as p e iously desc ibed
[131].
In i o ChEC and ChEC/2D analyses
Ch oma in endogenous clea age (ChEC) and ChEC/2D analyses o RFA1-MN cells we e
pe o med as epo ed [28]. B ie ly, cells g own unde he indica ed condi ions we e
a es ed wi h sodium azide (0.1% inal concen a ion). Fo clea age induc ion, cells
we e pe meabilized wi h digi onin and incuba ed wi h 2 mM CaCl2 a 30 °C unde gen le
agi a ion. Fo ChEC analyses, o al DNA was isola ed and esol ed in o 0.8% TAE 1×
aga ose gels. To analyse eplica ion in e media es (ChEC/2D), o al DNA was ex ac ed as
de ailed, diges ed wi h EcoRV and HindIII, esol ed by neu al/neu al wo-dimensional
(2D)-gel elec opho esis, blo ed o nylon memb anes, and analysed by hyb idiza ion wi h
he 32P-labelled p obe O . Signal was acqui ed in a Fuji FLA5100 wi h he ImageGauge
analysis p og am.
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 19 / 27
PLOS Gene icS B oken o k epai in yeas
Wes e n blo
Yeas p o ein ex ac s o analyse Rad53 phospho yla ion and R a1/R a1-MN exp ession we e
p epa ed using he TCA p o ocol [132]. P o ein samples we e esol ed by 8% SDS-PAGE,
p obed wi h an ibodies agains Rad53 (Abcam, ab104232), R a1 (Abcam, ab221198) o Pgk1
(In i ogen, 22C5D8) and de ec ed wi h a pe oxidase-conjuga e an ibody. The immunolu-
minescen signal was gene a ed wi h ei he he Wes e nB igh ECL (Ad ans a) o he Cla i y
Wes e n ECL Subs a e (BioRad) ki , acqui ed in a ChemiDoc MP image sys em and quan i-
ied wi h he Image Lab so wa e (Bio ad).
Suppo ing in o ma ion
S1 Fig. Cha ac e iza ion o RFA1-MN cells. (A) ChEC analysis o exponen ially g owing
cells exp essing R a1-MN incuba ed in he absence o p esence o 0.005% MMS o 2 h. To al
DNA om cells pe meabilized and ea ed wi h 2 mM CaCl2 o di e en imes is shown
(le ). Addi ion o Ca2+ is equi ed o de ec ion o R a1-MN-diges ed DNA, as de e mined
by unning o al DNA o wild- ype and RFA1-MN cells g owing in he absence o p esence o
0.005% MMS o 2 h ( igh ). (B) Ionizing adia ion and zeocin sensi i i y o RFA1-MN cells, as
de e mined by spo ing 10- old se ial dilu ions o he same numbe o mid-log g owing cells.
Wild- ype and ad52∆ cells we e included as con ol. (C) Budding index and doubling ime
o wild- ype and RFA1-MN cells. The mean and s anda d de ia ion o h ee (budding index)
and wo (doubling ime) independen expe imen s a e shown. (D) E ec o he RFA1-MN
chime a in he iabili y o wild- ype and ad52∆ cells ans o med wi h he URA3-based
plasmid pMDL5 exp essing Rad52 om he GAL1 p omo e in he indica ed media, as de e -
mined by spo ing 10- old se ial dilu ions o he same numbe o mid-log g owing cells. The
le hali y o he RFA1-MN ad52∆ s ain was escued wi h he URA3-based plasmid pDML5,
which exp esses RAD52 om he galac ose-inducible GAL1 p omo e . This s ain is able o
g ow, e en hough slowly, unde glucose- ep essing condi ions; howe e , his is due o basal
exp ession om he GAL1 p omo e , as indica ed by he lack o g ow h in he p esence o
luo oo o ic acid (FOA) whe e only U a- cells a e able o g ow. (E) E ec o HU and calcium
in he iabili y o RFA1-MN cells. The ad52∆ s ain was included o show he equi emen o
HR o he epai o HU-induced DNA lesions. The analyses we e epea ed a leas wice wi h
simila esul s.
(EPS)
S2 Fig. HR and ch oma in assembly equi emen s o RFA1-MN cell iabili y in he
absence and p esence o calcium. (A, C, and D) E ec o he indica ed mu a ions in he
g ow h o RFA1-MN cells as de e mined by spo ing 10- old se ial dilu ions o he same num-
be o mid-log g owing cells on o SMM medium wi hou o wi h he indica ed concen a ions
o CaCl2. (B) DSB sensi i i y o 105∆ cells o HO-induced DSBs, as de e mined by spo ing
10- old se ial dilu ions o he same numbe o mid-log g owing cells. Cells we e ans o med
wi h pGAL-HO and g own in glucose (GAL1p ep ession) and galac ose-con aining medium
(GAL1p ac i a ion). Wild- ype and ad52∆ cells we e included as con ol. The analyses we e
epea ed a leas wice wi h simila esul s. Mu an s sco ed in he SGA sc eening a e shown in
bold.
(EPS)
S3 Fig. Checkpoin ac i a ion acili a es he epai o b oken o ks. (A) Addi i e e ec
o dun1∆ and HU in he g ow h o RFA1-MN cells as de e mined by spo ing 10- old se ial
dilu ions o he same numbe o mid-log g owing cells on o SMM medium wi hou o wi h
he indica ed concen a ions o CaCl2 and HU. (B) Cell cycle p og ession and budding index
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 20 / 27
PLOS Gene icS B oken o k epai in yeas
o wild- ype cells synch onised in G1 wi h α- ac o and eleased in o S phase in he p esence
o 400mM CaCl2. The mean and s anda d de ia ion o h ee independen expe imen s a e
shown. (C) Rad53 ac i a ion in wild- ype cells ea ed o no wi h 0.005% MMS o 2 hou s in
he absence and p esence o 400mM CaCl2.
(EPS)
S4 Fig. Addi ional gene ic equi emen s o RFA1-MN cell iabili y in he absence and
p esence o calcium. (A–D) E ec o he indica ed mu a ions in he g ow h o RFA1-MN cells
as de e mined by spo ing 10- old se ial dilu ions o he same numbe o mid-log g owing
cells on o SMM medium wi hou o wi h he indica ed concen a ions o CaCl2. The analyses
we e epea ed a leas wice wi h simila esul s. Mu an s sco ed in he SGA sc eening a e
shown in bold.
(EPS)
S5 Fig. Raw da a o igu e panels. O iginal blo s o he indica ed igu e panels a e shown.
(EPS)
S1 Table. Saccha omyces ce e isiae genes s udied in he SGA sc eening. Genes analyzed in
he cus omized lib a y o null mu an s a e shown.
(XLSX)
S2 Table. Posi i e hi s om he SGA sc eening. The name o he posi i e hi s, hei PCR
alida ion and he e ec o he null mu an on RFA1-MN iabili y a e indica ed.
(XLSX)
S3 Table. Saccha omyces ce e isiae s ains used in his s udy. S ains, geno ypes and e e -
ences a e indica ed.
(DOCX)
S4 Table. Raw da a o igu e plo s. Raw alues o build budding index and doubling ime
plo s a e shown.
(XLSX)
Acknowledgmen s
We hank A u o Calzada, Ralph E. Wellinge , Mónica Segu ado and Ped o San Segundo o
a ious s ains and eagen s.
Au ho con ibu ions
Concep ualiza ion: Félix P ado.
Da a cu a ion: Ana Amiama-Roig.
Fo mal analysis: Ana Amiama-Roig.
Funding acquisi ion: Félix P ado.
In es iga ion: Ana Amiama-Roig, Ma a Ba ien os-Mo eno, Es he C uz-Zamb ano, Luz M
López-Ruiz, Román González-P ie o, Gab iel Rios-O elogio.
P ojec adminis a ion: Félix P ado.
Resou ces: Félix P ado.
Supe ision: Félix P ado.
Valida ion: Ana Amiama-Roig, Ma a Ba ien os-Mo eno, Es he C uz-Zamb ano, Luz M
López-Ruiz, Román González-P ie o, Gab iel Rios-O elogio.
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 21 / 27
PLOS Gene icS B oken o k epai in yeas
Visualiza ion: Ana Amiama-Roig, Ma a Ba ien os-Mo eno, Félix P ado.
W i ing – o iginal d a : Félix P ado.
W i ing – e iew & edi ing: Félix P ado.
Re e ences
1. Gailla d H, Ga cía-Muse T, Aguile a A. Replica ion s ess and cance . Na Re Cance .
2015;15(5):276–89. h ps://doi.o g/10.1038/n c3916 PMID: 25907220
2. Be i M, Co ez D, Lopes M. The plas ici y o DNA eplica ion o ks in esponse o clinically ele an
geno oxic s ess. Na Re Mol Cell Biol. 2020;21(10):633–51. h ps://doi.o g/10.1038/s41580-020-
0257-5 PMID: 32612242
3. P ado F. Homologous ecombina ion: o o k and beyond. Genes (Basel). 2018;9(12):603. h ps://doi.
o g/10.3390/genes9120603 PMID: 30518053
4. Kha ib JB, Nicolae CM, Moldo an G-L. Role o anslesion DNA syn hesis in he me abolism o
eplica ion-associa ed nascen s and gaps. J Mol Biol. 2024;436(1):168275. h ps://doi.o g/10.1016/j.
jmb.2023.168275 PMID: 37714300
5. Galan i L, P ande B. Righ ime, igh place-DNA damage and DNA eplica ion checkpoin s collec-
i ely sa egua d S phase. EMBO J. 2018;37(21):e100681. h ps://doi.o g/10.15252/embj.2018100681
PMID: 30287420
6. Chapman JR, Taylo MRG, Boul on SJ. Playing he end game: DNA double-s and b eak epai
pa hway choice. Mol Cell. 2012;47(4):497–510. h ps://doi.o g/10.1016/j.molcel.2012.07.029 PMID:
22920291
7. Scully R, Panday A, Elango R, Willis NA. DNA double-s and b eak epai -pa hway choice in soma ic
mammalian cells. Na Re Mol Cell Biol. 2019;20(11):698–714. h ps://doi.o g/10.1038/s41580-019-
0152-0 PMID: 31263220
8. Pâques F, Habe JE. Mul iple pa hways o ecombina ion induced by double-s and b eaks in
Saccha omyces ce e isiae. Mic obiol Mol Biol Re . 1999;63(2):349–404. h ps://doi.o g/10.1128/
MMBR.63.2.349-404.1999 PMID: 10357855
9. Wa e man DP, Habe JE, Smolka MB. Checkpoin esponses o DNA double-s and b eaks. Annu Re
Biochem. 2020;89:103–33. h ps://doi.o g/10.1146/annu e -biochem-011520-104722 PMID: 32176524
10. Liu L, Malko a A. B eak-induced eplica ion: un a eling each s ep. T ends Gene . 2022;38(7):752–65.
h ps://doi.o g/10.1016/j. ig.2022.03.011 PMID: 35459559
11. Wilson MA, Kwon Y, Xu Y, Chung W-H, Chi P, Niu H, e al. Pi 1 helicase and Polδ p omo e
ecombina ion-coupled DNA syn hesis ia bubble mig a ion. Na u e. 2013;502(7471):393–6. h ps://
doi.o g/10.1038/na u e12585 PMID: 24025768
12 . Deem A, Kesz helyi A, Blackg o e T, Vayl A, Co ey B, Ma hu R, e al. B eak-induced eplica ion is
highly inaccu a e. PLoS Biol. 2011;9(2):e1000594. h ps://doi.o g/10.1371/jou nal.pbio.1000594 PMID:
21347245
13 . Smi h CE, Llo en e B, Syming on LS. Templa e swi ching du ing b eak-induced eplica ion. Na u e.
2007;447(7140):102–5. h ps://doi.o g/10.1038/na u e05723 PMID: 17410126
14 . Malko a A, I ano EL, Habe JE. Double-s and b eak epai in he absence o RAD51 in yeas : a
possible ole o b eak-induced DNA eplica ion. P oc Na l Acad Sci U S A. 1996;93(14):7131–6.
h ps://doi.o g/10.1073/pnas.93.14.7131 PMID: 8692957
15 . Clemen e-Ruiz M, P ado F. Ch oma in assembly con ols eplica ion o k s abili y. EMBO Rep.
2009;10(7):790–6. h ps://doi.o g/10.1038/embo .2009.67 PMID: 19465889
16 . González-Ba e a S, Co és-Ledesma F, Wellinge RE, Aguile a A. Equal sis e ch oma id exchange is
a majo mechanism o double-s and b eak epai in yeas . Mol Cell. 2003;11(6):1661–71. h ps://doi.
o g/10.1016/s1097-2765(03)00183-7 PMID: 12820977
1 7. Mayle R, Campbell IM, Beck CR, Yu Y, Wilson M, Shaw CA, e al. DNA REPAIR. Mus81 and con e g-
ing o ks limi he mu agenici y o eplica ion o k b eakage. Science. 2015;349(6249):742–7. h ps://
doi.o g/10.1126/science.aaa8391 PMID: 26273056
18 . S umbe g D, Pilon AA, Smi h M, Hickey R, Malkas L, Pommie Y. Con e sion o opoisome ase I
clea age complexes on he leading s and o ibosomal DNA in o 5’-phospho yla ed DNA
double-s and b eaks by eplica ion uno . Mol Cell Biol. 2000;20(11):3977–87. h ps://doi.o g/10.1128/
MCB.20.11.3977-3987.2000 PMID: 10805740
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 22 / 27
PLOS Gene icS B oken o k epai in yeas
19 . V is KB, Dewa JM, Chis ol G, Wu RA, G aham TGW, Wal e JC. Single-s and DNA b eaks cause
eplisome disassembly. Mol Cell. 2021;81(6):1309–18.e6. h ps://doi.o g/10.1016/j.molcel.2020.12.039
PMID: 33484638
20. Pa ani R, T ipa hi V, V is KB, Zong D, Cha i R, Callen E, e al. S uc u e and epai o eplica ion-
coupled DNA b eaks. Science. 2024;385(6710):eado3867. h ps://doi.o g/10.1126/science.ado3867
PMID: 38900911
21. Kimble MT, Sane A, Reid RJD, Johnson MJ, Ro hs ein R, Syming on LS. Repai o eplica ion-
dependen double-s and b eaks di e s be ween he leading and lagging s ands. Mol Cell.
2025;85(1):61–77.e6. h ps://doi.o g/10.1016/j.molcel.2024.10.032 PMID: 39631395
22. Elango R, Nila a NM, Li AG, Nguyen D, Rass E, Du ey EE, e al. Two-ended ecombina ion a a
Flp-nickase-b oken eplica ion o k. Mol Cell. 2025;85(1):78–90.e3. h ps://doi.o g/10.1016/j.mol-
cel.2024.11.006 PMID: 39631396
23. Xu Y, Mo ow CA, Laksi Y, Hol OM, Taylo K, Tsiappou dhi C, e al. DNA nicks in bo h leading and
lagging s and empla es can igge b eak-induced eplica ion. Mol Cell. 2025;85(1):91-106.e5. h ps://
doi.o g/10.1016/j.molcel.2024.10.026 PMID: 39561776
24. Mache e M, Halazone is TD. DNA eplica ion s ess as a hallma k o cance . Annu Re Pa hol.
2015;10:425–48. h ps://doi.o g/10.1146/annu e -pa hol-012414-040424 PMID: 25621662
25. Hashimo o Y, Puddu F, Cos anzo V. RAD51- and MRE11-dependen eassembly o uncoupled CMG
helicase complex a collapsed eplica ion o ks. Na S uc Mol Biol. 2011;19(1):17–24. h ps://doi.
o g/10.1038/nsmb.2177 PMID: 22139015
26. Schmid M, Du ussel T, Laemmli UK. ChIC and ChEC; genomic mapping o ch oma in p o eins. Mol
Cell. 2004;16(1):147–57. h ps://doi.o g/10.1016/j.molcel.2004.09.007 PMID: 15469830
2 7. González-P ie o R, Muñoz-Cabello AM, Cabello-Loba o MJ, P ado F. Rad51 eplica ion o k ec ui -
men is equi ed o DNA damage ole ance. EMBO J. 2013;32(9):1307–21. h ps://doi.o g/10.1038/
emboj.2013.73 PMID: 23563117
28. González-P ie o R, Cabello-Loba o MJ, P ado F. In Vi o Binding o Recombina ion P o eins o
Non-DSB DNA Lesions and o Replica ion Fo ks. Me hods Mol Biol. 2021;2153:447–58. h ps://doi.
o g/10.1007/978-1-0716-0644-5_31 PMID: 32840798
29. Cano-Lina es MI, Yáñez-Vilches A, Ga cía-Rod íguez N, Ba ien os-Mo eno M, González-P ie o R,
San-Segundo P, e al. Non- ecombinogenic oles o Rad52 in anslesion syn hesis du ing DNA
damage ole ance. EMBO Rep. 2021;22(1):e50410. h ps://doi.o g/10.15252/emb .202050410 PMID:
33289333
30. Cabello-Loba o MJ, González-Ga ido C, Cano-Lina es MI, Wong RP, Yáñez-Vílchez A, Mo illo-
Huesca M, e al. Physical in e ac ions be ween MCM and Rad51 acili a e eplica ion o k lesion
bypass and ssDNA gap illing by non- ecombinogenic unc ions. Cell Rep. 2021;36(4):109440. h ps://
doi.o g/10.1016/j.cel ep.2021.109440 PMID: 34320356
31. González-Ga ido C, P ado F. Pa en al his one dis ibu ion and loca ion o he eplica ion obs acle
a nascen s ands con ol homologous ecombina ion. Cell Rep. 2023;42(3):112174. h ps://doi.
o g/10.1016/j.cel ep.2023.112174 PMID: 36862554
32. Hung S-H, Wong RP, Ul ich HD, Kao C-F. Monoubiqui yla ion o his one H2B con ibu es
o he bypass o DNA damage du ing and a e DNA eplica ion. P oc Na l Acad Sci U S A.
2017;114(11):E2205–14. h ps://doi.o g/10.1073/pnas.1612633114 PMID: 28246327
33. Li win I, Bakowski T, Szakal B, Pila czyk E, Maciaszczyk-Dziubinska E, B anzei D, e al. E o - ee
DNA damage ole ance pa hway is acili a ed by he I c5 anslocase h ough cohesin. EMBO J.
2018;37(18):e98732. h ps://doi.o g/10.15252/embj.201798732 PMID: 30111537
34. Due a R, Iliakis G. Replica ion p o ein A: a mul i unc ional p o ein wi h oles in DNA eplica ion,
epai and beyond. NAR Cance . 2020;2(3):zcaa022. h ps://doi.o g/10.1093/na can/zcaa022 PMID:
34316690
35. Zhang S, Wang X, Zhao H, Shi J, Chen X. New insigh s in o he mechanism o RPA in p e-
se ing genome s abili y. GENOME INSTAB DIS. 2022;3(5):255–66. h ps://doi.o g/10.1007/
s42764-022-00085-y
36. Siko ski TW, Fica o SB, Holik J, Kim T, Rando OJ, Ma o JA, e al. Sub1 and RPA associa e wi h
RNA polyme ase II a di e en s ages o ansc ip ion. Mol Cell. 2011;44(3):397–409. h ps://doi.
o g/10.1016/j.molcel.2011.09.013 PMID: 22055186
3 7. Li S, Xu Z, Xu J, Zuo L, Yu C, Zheng P, e al. R 105 unc ions as a chape one o eplica ion p o ein A
o p ese e genome s abili y. EMBO J. 2018;37(17):e99154. h ps://doi.o g/10.15252/embj.201899154
PMID: 30065069
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 23 / 27
PLOS Gene icS B oken o k epai in yeas
38. Li L, Wang J, Yang Z, Zhao Y, Jiang H, Jiang L, e al. Me abolic emodeling main ains a educing en i-
onmen o apid ac i a ion o he yeas DNA eplica ion checkpoin . EMBO J. 2022;41(4):e108290.
h ps://doi.o g/10.15252/embj.2021108290 PMID: 35028974
39. Wong RP, Ga cía-Rod íguez N, Zilio N, Hanulo á M, Ul ich HD. P ocessing o DNA
Polyme ase-Blocking Lesions du ing Genome Replica ion Is Spa ially and Tempo ally Seg ega ed
om Replica ion Fo ks. Mol Cell. 2020;77(1):3–16.e4. h ps://doi.o g/10.1016/j.molcel.2019.09.015
PMID: 31607544
40. K ame KM, B ock JA, Bloom K, Moo e JK, Habe JE. Two di e en ypes o double-s and b eaks in
Saccha omyces ce e isiae a e epai ed by simila RAD52-independen , nonhomologous ecombi-
na ion e en s. Mol Cell Biol. 1994;14(2):1293–301. h ps://doi.o g/10.1128/mcb.14.2.1293-1301.1994
PMID: 8289808
41. Wya HDM, Wes SC. Holliday junc ion esol ases. Cold Sp ing Ha b Pe spec Biol.
2014;6(9):a023192. h ps://doi.o g/10.1101/cshpe spec .a023192 PMID: 25183833
42. Kuzmin E, Cos anzo M, And ews B, Boone C. Syn he ic Gene ic A ay Analysis. Cold Sp ing Ha b
P o oc. 2016;2016(4):pdb.p o 088807. h ps://doi.o g/10.1101/pdb.p o 088807 PMID: 27037072
43. Kelle maye R, Aiello DP, Mise a A, Bedwell DM. Ex acellula Ca(2+) sensing con ibu es o excess
Ca(2+) accumula ion and acuola agmen a ion in a pm 1Del a mu an o S. ce e isiae. J Cell Sci.
2003;116(P 8):1637–46. h ps://doi.o g/10.1242/jcs.00372 PMID: 12640047
44. San Filippo J, Sung P, Klein H. Mechanism o euka yo ic homologous ecombina ion. Annu Re Bio-
chem. 2008;77:229–57. h ps://doi.o g/10.1146/annu e .biochem.77.061306.125255 PMID: 18275380
45. P ado F, Co és-Ledesma F, Hue as P, Aguile a A. Mi o ic ecombina ion in Saccha omyces ce e i-
siae. Cu Gene . 2003;42(4):185–98. h ps://doi.o g/10.1007/s00294-002-0346-3 PMID: 12589470
46. Wang X, Dong Y, Zhao X, Li J, Lee J, Yan Z, e al. R 105 p omo es high- ideli y DNA eplica ion
and epai by egula ing he single-s anded DNA-binding ac o RPA. P oc Na l Acad Sci U S A.
2021;118(25):e2106393118. h ps://doi.o g/10.1073/pnas.2106393118 PMID: 34140406
4 7. Ru P, Donnianni RA, Glancy E, Oh J, Syming on LS. RPA s abiliza ion o single-s anded DNA is
c i ical o b eak-induced eplica ion. Cell Rep. 2016;17(12):3359–68. h ps://doi.o g/10.1016/j.cel-
ep.2016.12.003 PMID: 28009302
48. Syming on LS. Mechanism and egula ion o DNA end esec ion in euka yo es. C i Re Biochem Mol
Biol. 2016;51(3):195–212. h ps://doi.o g/10.3109/10409238.2016.1172552 PMID: 27098756
49. I a G, Malko a A, Libe i G, Foiani M, Habe JE. S s2 and Sgs1-Top3 supp ess c osso e s du ing
double-s and b eak epai in yeas . Cell. 2003;115(4):401–11. h ps://doi.o g/10.1016/s0092-
8674(03)00886-9 PMID: 14622595
50. Ip SCY, Rass U, Blanco MG, Flynn HR, Skehel JM, Wes SC. Iden i ica ion o Holliday junc ion esol-
ases om humans and yeas . Na u e. 2008;456(7220):357–61. h ps://doi.o g/10.1038/na u e07470
PMID: 19020614
51. Godin S, Wie A, Kabbina a F, B a on-Palme DS, Ghodke H, Van Hou en B, e al. The Shu complex
in e ac s wi h Rad51 h ough he Rad51 pa alogues Rad55-Rad57 o media e e o - ee ecombina-
ion. Nucleic Acids Res. 2013;41(8):4525–34. h ps://doi.o g/10.1093/na /gk 138 PMID: 23460207
52. Godin SK, Zhang Z, He ken BW, Wes mo eland JW, Lee AG, Mihale ic MJ, e al. The Shu complex
p omo es e o - ee ole ance o alkyla ion-induced base excision epai p oduc s. Nucleic Acids Res.
2016;44(17):8199–215. h ps://doi.o g/10.1093/na /gkw535 PMID: 27298254
53. Sho E, Weins ein J, Ro hs ein R. A gene ic sc een o op3 supp esso s in Saccha omyces ce e i-
siae iden i ies SHU1, SHU2, PSY3 and CSM2: ou genes in ol ed in e o - ee DNA epai . Gene ics.
2005;169(3):1275–89. h ps://doi.o g/10.1534/gene ics.104.036764 PMID: 15654096
54. Collins SR, Mille KM, Maas NL, Rogue A, Fillingham J, Chu CS, e al. Func ional dissec ion o
p o ein complexes in ol ed in yeas ch omosome biology using a gene ic in e ac ion map. Na u e.
2007;446(7137):806–10. h ps://doi.o g/10.1038/na u e05649 PMID: 17314980
55. Pan X, Ye P, Yuan DS, Wang X, Bade JS, Boeke JD. A DNA in eg i y ne wo k in he yeas Saccha-
omyces ce e isiae. Cell. 2006;124(5):1069–81. h ps://doi.o g/10.1016/j.cell.2005.12.036 PMID:
16487579
56. D iscoll R, Hudson A, Jackson SP. Yeas R 109 p omo es genome s abili y by ace yla ing his one
H3 on lysine 56. Science. 2007;315(5812):649–52. h ps://doi.o g/10.1126/science.1135862 PMID:
17272722
5 7. Han J, Zhou H, Ho azdo sky B, Zhang K, Xu R-M, Zhang Z. R 109 ace yla es his one H3 lysine
56 and unc ions in DNA eplica ion. Science. 2007;315(5812):653–5. h ps://doi.o g/10.1126/sci-
ence.1133234 PMID: 17272723
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 24 / 27
PLOS Gene icS B oken o k epai in yeas
58. Li Q, Zhou H, Wu ele H, Da ies B, Ho azdo sky B, Ve eaul A, e al. Ace yla ion o his one H3
lysine 56 egula es eplica ion-coupled nucleosome assembly. Cell. 2008;134(2):244–55. h ps://doi.
o g/10.1016/j.cell.2008.06.018 PMID: 18662540
59. Han J, Zhang H, Zhang H, Wang Z, Zhou H, Zhang Z. A Cul4 E3 ubiqui in ligase egula es his-
one hand-o du ing nucleosome assembly. Cell. 2013;155(4):817–29. h ps://doi.o g/10.1016/j.
cell.2013.10.014 PMID: 24209620
60. Buse R, Kellne V, Melnik A, Wilson-Zbinden C, Schellhaas R, Kas ne L, e al. The Replisome-
Coupled E3 Ubiqui in Ligase R 101Mms22 Coun e ac s M c1 Func ion o Tole a e Geno oxic S ess.
PLoS Gene . 2016;12(2):e1005843. h ps://doi.o g/10.1371/jou nal.pgen.1005843 PMID: 26849847
61. P ado F, Co és-Ledesma F, Aguile a A. The absence o he yeas ch oma in assembly ac o As 1
inc eases genomic ins abili y and sis e ch oma id exchange. EMBO Rep. 2004;5(5):497–502. h ps://
doi.o g/10.1038/sj.embo .7400128 PMID: 15071494
62. Luke B, Ve sini G, Jaquenoud M, Zaidi IW, Ku z T, Pin a d L, e al. The cullin R 101p p omo es epli-
ca ion o k p og ession h ough damaged DNA and na u al pause si es. Cu Biol. 2006;16(8):786–92.
h ps://doi.o g/10.1016/j.cub.2006.02.071 PMID: 16631586
63. Du o E, Vaisica JA, B own GW, Rouse J. Budding yeas Mms22 and Mms1 egula e homologous
ecombina ion induced by eplisome blockage. DNA Repai (Ams ). 2008;7(5):811–8. h ps://doi.
o g/10.1016/j.dna ep.2008.01.007 PMID: 18321796
64. Wu ele H, Kaise GS, Bacal J, S -Hilai e E, Lee E-H, Tsao S, e al. His one H3 lysine 56 ace yla-
ion and he esponse o DNA eplica ion o k damage. Mol Cell Biol. 2012;32(1):154–72. h ps://doi.
o g/10.1128/MCB.05415-11 PMID: 22025679
65. Clemen e-Ruiz M, González-P ie o R, P ado F. His one H3K56 ace yla ion, CAF1, and R 106
coo dina e nucleosome assembly and s abili y o ad ancing eplica ion o ks. PLoS Gene .
2011;7(11):e1002376. h ps://doi.o g/10.1371/jou nal.pgen.1002376 PMID: 22102830
66. Luciano P, Dehé P-M, Audebe S, Géli V, Co da Y. Replisome unc ion du ing eplica i e s ess is
modula ed by his one h3 lysine 56 ace yla ion h ough C 4. Gene ics. 2015;199(4):1047–63. h ps://
doi.o g/10.1534/gene ics.114.173856 PMID: 25697176
6 7. E kmann JA, Kau man PD. A nega i ely cha ged esidue in place o his one H3K56 suppo s
ch oma in assembly ac o associa ion bu no geno oxic s ess esis ance. DNA Repai (Ams ).
2009;8(12):1371–9. h ps://doi.o g/10.1016/j.dna ep.2009.09.004 PMID: 19796999
68. Yang J, Zhang X, Feng J, Leng H, Li S, Xiao J, e al. The His one Chape one FACT Con ibu es
o DNA Replica ion-Coupled Nucleosome Assembly. Cell Rep. 2016;14(5):1128–41. h ps://doi.
o g/10.1016/j.cel ep.2015.12.096 PMID: 26804921
69. Gan H, Se a-Ca dona A, Hua X, Zhou H, Labib K, Yu C, e al. The Mcm2-C 4-Polα Axis Facili a es
Pa en al His one H3-H4 T ans e o Lagging S ands. Mol Cell. 2018;72(1):140–51.e3. h ps://doi.
o g/10.1016/j.molcel.2018.09.001 PMID: 30244834
70. Li Z, Hua X, Se a-Ca dona A, Xu X, Gan S, Zhou H, e al. DNA polyme ase α in e ac s wi h H3-H4
and acili a es he ans e o pa en al his ones o lagging s ands. Sci Ad . 2020;6(35):eabb5820.
h ps://doi.o g/10.1126/sciad .abb5820 PMID: 32923642
71. Yu C, Gan H, Se a-Ca dona A, Zhang L, Gan S, Sha ma S, e al. A mechanism o p e en ing asym-
me ic his one seg ega ion on o eplica ing DNA s ands. Science. 2018;361(6409):1386–9. h ps://doi.
o g/10.1126/science.aa 8849 PMID: 30115745
72. Maas NL, Mille KM, DeFazio LG, Toczyski DP. Cell cycle and checkpoin egula ion o his one
H3 K56 ace yla ion by Hs 3 and Hs 4. Mol Cell. 2006;23(1):109–19. h ps://doi.o g/10.1016/j.mol-
cel.2006.06.006 PMID: 16818235
73. Celic I, Masumo o H, G i i h WP, Meluh P, Co e RJ, Boeke JD, e al. The si uins hs 3 and Hs 4p p e-
se e genome in eg i y by con olling his one h3 lysine 56 deace yla ion. Cu Biol. 2006;16(13):1280–
9. h ps://doi.o g/10.1016/j.cub.2006.06.023 PMID: 16815704
74. Che J, Smi h S, Kim YJ, Shim EY, Myung K, Lee SE. Hype -Ace yla ion o His one H3K56
Limi s B eak-Induced Replica ion by Inhibi ing Ex ensi e Repai Syn hesis. PLoS Gene .
2015;11(2):e1004990. h ps://doi.o g/10.1371/jou nal.pgen.1004990 PMID: 25705897
75. Robe s TM, Zaidi IW, Vaisica JA, Pe e M, B own GW. Regula ion o 107 ec ui men o s alled DNA
eplica ion o ks by he cullin 101 and he 109 ace yl ans e ase. Mol Biol Cell. 2008;19(1):171–80.
h ps://doi.o g/10.1091/mbc.e07-09-0961 PMID: 17978089
76. Hang LE, Peng J, Tan W, Szakal B, Menol i D, Sheng Z, e al. R 107 Is a Mul i- unc ional Sca -
old Suppo ing Replica ion P og ession wi h Pa ne SUMO and Ubiqui in Ligases. Mol Cell.
2015;60(2):268–79. h ps://doi.o g/10.1016/j.molcel.2015.08.023 PMID: 26439300
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011405 Ap il 1, 2025 25 / 27
PLOS Gene icS B oken o k epai in yeas
7 7. P incz LN, Wild P, Bi mann J, Aguado FJ, Blanco MG, Ma os J, e al. Db 4-dependen kinase and he
R 107 sca old p omo e Mus81-Mms4 esol ase ac i a ion du ing mi osis. EMBO J. 2017;36(5):664–
78. h ps://doi.o g/10.15252/embj.201694831 PMID: 28096179
78. Be múdez-López M, Villo ia MT, Es e as M, Ja muz A, To es-Rosell J, Clemen e-Blanco A, e al.
Sgs1’s oles in DNA end esec ion, HJ dissolu ion, and c osso e supp ession equi e a wo-s ep
SUMO egula ion dependen on Smc5/6. Genes De . 2016;30(11):1339–56. h ps://doi.o g/10.1101/
gad.278275.116 PMID: 27298337
79. B anzei D, Vanoli F, Foiani M. SUMOyla ion egula es Rad18-media ed empla e swi ch. Na u e.
2008;456(7224):915–20. h ps://doi.o g/10.1038/na u e07587 PMID: 19092928
80. Hoege C, P ande B, Moldo an G-L, Py owolakis G, Jen sch S. RAD6-dependen DNA epai is
linked o modi ica ion o PCNA by ubiqui in and SUMO. Na u e. 2002;419(6903):135–41. h ps://doi.
o g/10.1038/na u e00991 PMID: 12226657
81. Kang M-J, Lee C-H, Kang Y-H, Cho I-T, Nguyen TA, Seo Y-S. Gene ic and unc ional in e ac ions
be ween Mus81-Mms4 and Rad27. Nucleic Acids Res. 2010;38(21):7611–25. h ps://doi.o g/10.1093/
na /gkq651 PMID: 20660481
82. Ha ison JC, Habe JE. Su i ing he b eakup: he DNA damage checkpoin . Annu Re Gene .
2006;40:209–35. h ps://doi.o g/10.1146/annu e .gene .40.051206.105231 PMID: 16805667
83. Jossen R, Be mejo R. The DNA damage checkpoin esponse o eplica ion s ess: A Game o Fo ks.
F on Gene . 2013;4:26. h ps://doi.o g/10.3389/ gene.2013.00026 PMID: 23493417
84. Fe a i M, Dibi e o D, De G ego io G, Eapen VV, Rawal CC, Lazza o F, e al. Func ional in e play
be ween he 53BP1-o holog Rad9 and he M e11 complex egula es esec ion, end- e he ing and
epai o a double-s and b eak. PLoS Gene . 2015;11(1):e1004928. h ps://doi.o g/10.1371/jou nal.
pgen.1004928 PMID: 25569305
85. Bu gess RJ, Han J, Zhang Z. The Ddc1-Mec3-Rad17 sliding clamp egula es his one-his one chape -
one in e ac ions and DNA eplica ion-coupled nucleosome assembly in budding yeas . J Biol Chem.
2014;289(15):10518–29. h ps://doi.o g/10.1074/jbc.M114.552463 PMID: 24573675
86. Ka as GI, Fumasoni M, Sienski G, Vanoli F, B anzei D, Jen sch S. Noncanonical ole o he 9-1-1
clamp in he e o - ee DNA damage ole ance pa hway. Mol Cell. 2013;49(3):536–46. h ps://doi.
o g/10.1016/j.molcel.2012.11.016 PMID: 23260657
8 7. Lou H, Koma a M, Ka ou Y, Guan Z, Reis CC, Budd M, e al. M c1 and DNA polyme ase epsilon
unc ion oge he in linking DNA eplica ion and he S phase checkpoin . Mol Cell. 2008;32(1):106–17.
h ps://doi.o g/10.1016/j.molcel.2008.08.020 PMID: 18851837
88. Ka ou Y, Kanoh Y, Bando M, Noguchi H, Tanaka H, Ashika i T, e al. S-phase checkpoin p o eins To 1
and M c1 o m a s able eplica ion-pausing complex. Na u e. 2003;424(6952):1078–83. h ps://doi.
o g/10.1038/na u e01900 PMID: 12944972
89. Calzada A, Hodgson B, Kanemaki M, Bueno A, Labib K. Molecula ana omy and egula ion o a s able
eplisome a a paused euka yo ic DNA eplica ion o k. Genes De . 2005;19(16):1905–19. h ps://doi.
o g/10.1101/gad.337205 PMID: 16103218
90. S ini asan M, Fumasoni M, Pe ela NJ, Mu ay A, Nasmy h KA. Cohesion is es ablished du ing DNA
eplica ion u ilising ch omosome associa ed cohesin ings as well as hose loaded de no o on o
nascen DNAs. Eli e. 2020;9:e56611. h ps://doi.o g/10.7554/eLi e.56611 PMID: 32515737
91. Tou iè e H, Ve sini G, Co dón-P eciado V, Alabe C, Pase o P. M c1 and To 1 p omo e eplica ion
o k p og ession and eco e y independen ly o Rad53. Mol Cell. 2005;19(5):699–706. h ps://doi.
o g/10.1016/j.molcel.2005.07.028 PMID: 16137625
92. C abbé L, Thomas A, Pan esco V, De Vos J, Pase o P, Leng onne A. Analysis o eplica ion p o-
iles e eals key ole o RFC-C 18 in yeas eplica ion s ess esponse. Na S uc Mol Biol.
2010;17(11):1391–7. h ps://doi.o g/10.1038/nsmb.1932 PMID: 20972444
93. Leng onne A, McIn y e J, Ka ou Y, Kanoh Y, Hop ne K-P, Shi ahige K, e al. Es ablishmen o sis e
ch oma id cohesion a he S. ce e isiae eplica ion o k. Mol Cell. 2006;23(6):787–99. h ps://doi.
o g/10.1016/j.molcel.2006.08.018 PMID: 16962805
94. Chang M, Bellaoui M, Boone C, B own GW. A genome-wide sc een o me hyl me hanesul ona e-
sensi i e mu an s e eals genes equi ed o S phase p og ession in he p esence o DNA damage.
P oc Na l Acad Sci U S A. 2002;99(26):16934–9. h ps://doi.o g/10.1073/pnas.262669299 PMID:
12482937
95. Hoch NC, Chen ES-W, Buckland R, Wang S-C, Fazio A, Hamme A, e al. Molecula basis o he
essen ial s phase unc ion o he ad53 checkpoin kinase. Mol Cell Biol. 2013;33(16):3202–13. h ps://
doi.o g/10.1128/MCB.00474-13 PMID: 23754745
