MINI REVIEW
published: 10 Janua y 2020
doi: 10.3389/ molb.2019.00153
F on ie s in Molecula Biosciences | www. on ie sin.o g 1Janua y 2020 | Volume 6 | A icle 153
Edi ed by:
Se gio Fe nandes De Almeida,
Facul y o Medicine, Uni e si y o
Lisbon, Po ugal
Re iewed by:
Law ence Po i k,
Vi ginia Commonweal h Uni e si y,
Uni ed S a es
Ka ja Lammens,
Ludwig Maximilian Uni e si y o
Munich, Ge many
*Co espondence:
Felipe Co és-Ledesma
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Cellula Biochemis y,
a sec ion o he jou nal
F on ie s in Molecula Biosciences
Recei ed: 10 Oc obe 2019
Accep ed: 11 Decembe 2019
Published: 10 Janua y 2020
Ci a ion:
Se ano-Bení ez A, Co és-Ledesma F
and Ruiz JF (2020) “An End o a
Means”: How DNA-End S uc u e
Shapes he Double-S and B eak
Repai P ocess.
F on . Mol. Biosci. 6:153.
doi: 10.3389/ molb.2019.00153
“An End o a Means”: How DNA-End
S uc u e Shapes he Double-S and
B eak Repai P ocess
Almudena Se ano-Bení ez1, Felipe Co és-Ledesma1,2*and Jose F. Ruiz1,3
1Andalusian Cen e o Molecula Biology and Regene a i e Medicine (CABIMER-CSIC-Uni e si y o Se ille-Pablo de Ola ide
Uni e si y), Se ille, Spain, 2Topology and DNA b eaks G oup, Spanish Na ional Cance Resea ch Cen e , Mad id, Spain,
3Depa men o Plan Biochemis y and Molecula Biology, Uni e si y o Se ille, Se ille, Spain
Endogenously-a ising DNA double-s and b eaks (DSBs) a ely ha bo canonical
5′-phospha e, 3′-hyd oxyl moie ies a he ends, which a e, ega dless o he pa hway
used, ul ima ely equi ed o hei epai . Cells a e he e o e endowed wi h a wide a ie y
o enzymes ha can deal wi h hese chemical and s uc u al a ia ions and gua an ee he
o ma ion o liga able e mini. An impo an dis inc ion is whe he he ends a e di ec ly
“unblocked” by speci ic enzyma ic ac i i ies wi hou a ec ing he in eg i y o he DNA
molecule and i s sequence, o whe he hey a e “p ocessed” by unspeci ic nucleases
ha emo e nucleo ides om he e mini. DNA end s uc u e and con igu a ion, he e o e,
shape he epai p ocess, i s equi emen s, and, impo an ly, i s inal ou come. Thus, he
molecula mechanisms ha coo dina e and in eg a e he cellula esponse o blocked
DSBs, al hough s ill la gely unexplo ed, can be pa icula ly ele an o main aining
genome in eg i y and a oiding malignan ans o ma ion and cance .
Keywo ds: DNA double s and b eak (DSB), Non-homologous DNA end joining, ATM, DNA-PK ca aly ic subuni ,
genome ins abili y
Double-s and b eaks (DSBs) a e he mos de as a ing lesion ha DNA molecule can su e . Indeed,
hey can cause dange ous ch omosomal ea angemen s o e en cell dea h i hey a e no p ope ly
epai ed. In gene al e ms, he e a e wo concep ually di e en pa hways o epai DSBs ha can be
di ided in o hose ha use homologous sequences—ei he a sis e ch oma id o ano he sequence
elsewhe e in he genome—as a empla e in he epai (homologous ecombina ion, HR), and hose
ha di ec ly ejoin he ends, wi hou any empla e equi emen (Liebe , 2008; San Filippo e al.,
2008; Pannunzio e al., 2018), ega dless o whe he using minimal (non-homologous end-joining,
NHEJ) o mo e ex ensi e (mic ohomology-media ed end-joining, MMEJ) mic ohomologies o
s abilize he junc ions. Despi e he gene al in insic diploidy o soma ic mammalian cells, HR a ely
uses he homologous ch omosome as a empla e o DSB epai (Johnson, 2000). Consequen ly,
HR is mos ly es ic ed o la e S/G2 phase, when a sis e ch oma id is a ailable, whe eas NHEJ
can ope a e in any phase o he cell cycle. Besides his global dis inc ion, he e a e addi ional
peculia i ies o DSB epai mechanisms based on he speci ic na u e o each DNA lesion, speci ically
when i comes o he chemical con igu a ion o he b oken DNA ends. In his ega d, since he HR
will use he in o ma ion o an in ac empla e o epai (San Filippo e al., 2008), he ends o he
b eak, bo h 5’ and 3′, can be ex ensi ely deg aded wi hou comp omising an e icien econs i u ion
o he ini ially los DNA sequences. In con as , he chemical modi ica ions o DSB ends, and how
hese a e sol ed, a e pi o al in he NHEJ p ocess and inal epai ou come. I is he e o e o g ea
in e es o unde s and how DSBs ha bo ing complex DNA ends a e epai ed in he G1 phase o he
cell cycle, du ing which HR is s ongly limi ed.
Se ano-Bení ez e al. DNA-End S uc u e and DSB Repai
THE NHEJ PROCESS
The s a ing poin o he NHEJ p ocess akes place wi h he
ecogni ion and binding o double s anded DNA ends by
he KU70/80 he e odime , which occu s in an ex ao dina ily
e icien manne due o i s abundance and i s s ong a idi y o
his ype o DNA subs a e. DNA-bound KU he e odime , in
u n, ec ui s DNA-PKcs o o m he DNA-PK holoenzyme, so
ha he wo DNA-PKcs molecules bound o opposing sides o
he DSB can in e ac one each o he , con ibu ing o synapsis o
b oken DNA ends (Meek e al., 2008; Neal and Meek, 2011). The
DNA-PK complex is he main egula o o he NHEJ p ocess,
coo dina ing he ec ui men o downs eam NHEJ accesso y
ac o s, such as X- ay c oss complemen ing g oup 4 (XRCC4),
XRCC4-like ac o /Ce nunnos (XLF), o Pa alog o XRCC4 and
XLF (PAXX), and DNA ligase IV (LIG4), which con ibu e o
he p ope pai ing o DSB ends and pe o m he inal liga ion
o he b eak (Kaka ougkas and Jeggo, 2014; Ochi e al., 2015;
Conlin e al., 2017). In e eb a es, NHEJ u he e ol ed an end
p ocessing capaci y ha allows o he epai o complex ends
(e.g., hai pins), and which is also, in pa , egula ed by DNA-PK,
as will be discussed below.
RELEVANCE OF END STRUCTURE AND
CONFIGURATION DURING NHEJ
I can be claimed ha he only essen ial s ep o NHEJ
p ocess is he liga ion o one o he DNA s ands o he DSB
(Wa e s e al., 2014). Du ing his p ocess, LIG4 ac i i y equi es
compa ible ends ha bo ing canonical 5′-phospha e and 3′-
hyd oxyl e mini. Howe e , DSBs o en ha e complex ends wi h
chemical modi ica ions o s uc u es ha do no allow s aigh -
o wa d joining o he e mini, so hey can be conside ed as
blocked ends (Figu e 1). These chemical a ia ions can be sensed
by LIG4 h ough he dis up ion o i s ca aly ic cycle (Reid e al.,
2017). The e o e, when DSBs ha bo non-canonical chemical
s uc u es a he ends, hey mus be es o ed o con en ional
5′-phospha e and 3′-hyd oxyl e mini so ha DNA liga ion can
ake place. The e a e wo concep ually di e en ways by which
hese non-canonical DNA ends can be con e ed in o liga able
subs a es (Figu e 1). On he one hand, cells ha e a a ie y o
enzymes o di ec ly es o e he canonical chemical s uc u e.
Gi en ha his e en does no in ol e any sequence modi ica ion,
i can be simply conside ed as an “unblocking” p ocess. On he
o he hand, unde ce ain ci cums ances, such as he p esence
o complex lesions, unblocking ac i i ies may be comp omised
o o e whelmed, esul ing in DSBs ha equi e addi ional
“end p ocessing” by he ac ion o nucleases ha clea e DNA
sequence om he ends o emo e he chemical modi ica ions
(Figu e 1). Rega ding unblocking, he e is a la ge numbe o
ac o s wi h di e en enzyma ic ac i i ies ha a e a ailable
o his p ocess du ing NHEJ (Figu e 2), such as y osyl-DNA
phosphodies e ases 1 and 2 (TDP1 and TDP2, espec i ely),
polynucleo ide kinase (PNKP), Ap a axin, and e en KU. This,
in u n, e lec s he wide a ie y o damaged e mini ha
can a ise, as each o hese ac o s emo es speci ic chemical
modi ica ions a DNA ends (Po i k, 2012; And es e al., 2015).
These unblocking ac i i ies a e essen ial in NHEJ, since hey
a e esponsible o acili a ing accu a e eliga ion o he b eaks,
as opposed o he p ocessing o DNA ends ha may in ol e
nucleo ide loss o gain and, he e o e, sequence modi ica ion.
In e es ingly, ionizing adia ion, which is a common and well-
es ablished sou ce o DSBs, mos ly induces blocked e mini wi h
he e ogeneous end s uc u es. Damage occu s ei he di ec ly,
by high-ene gy pa icle collision wi h DNA, o indi ec ly, when
hese pa icles spli wa e molecules leading o dange ous ee
adicals; in bo h cases his mainly esul s in b eakage o he
suga backbone, and he e o e needs o be p ocessed, necessa ily
leading o loss o one nucleo ide om he e mini (Reisz e al.,
2014).
Ano he aspec o highligh in NHEJ is he possible
incompa ibili y among DSBs o be epai ed due o he absence
o sequence complemen a i y o DNA ends. This si ua ion may
occu when DSB ends ha e small p o ube ances, ei he wi h
5′o 3′pola i y. The sho s e ches o single s anded DNA
o hese o e hangs may be compa ible (ei he ully o pa ially
complemen a y sequences) o no . I has been shown ha LIG4
can liga e ac oss sho gaps o ejoin se e al incompa ible DNA
end con igu a ions ha do no sha e e en 1-bp o e minal
mic ohomology (Gu e al., 2007). Fo his scena ios, NHEJ also
akes ad an age o se e al p ocessing enzymes ha can modi y
DNA ends un il hey become liga able subs a es (S ande e al.,
2012). In his way, single-s anded DNA o e hangs (as also may
happen wi h blun ends) can be immed by nucleases such as
ARTEMIS gi ing ise o small gaps han can be e icien ly illed-in
by specialized X amily DNA polyme ases (see below) (Mahajan
e al., 2002; Lee J. W. e al., 2004; Ma e al., 2004; McElhinny e al.,
2005; Capp e al., 2007; Liebe , 2010). I is wo h no ing ha non-
complemen a y DNA ends a e indeed he mos likely esul o
end p ocessing a ini ially chemically modi ied s uc u es.
NHEJ: AN ITERATIVE VS A HIERARCHICAL
PROCESS
Al hough NHEJ is gene ally conside ed a single DNA epai
pa hway, a wide a ie y o ac o s a e needed and di e en sub-
ou es can be dis inguished depending on he di e en DSB end
con igu a ions (Pannunzio e al., 2018). In addi ion, he e is
s ill impo an deba e abou how hese NHEJ accesso y ac o s
ac ually ope a e, and, in his sense, wo appa en ly an agonis ic
posi ions can now be dis inguished. On he one hand, some
au ho s p opose ha NHEJ ac o s ope a e in an i e a i e way
wi hou an es ablished o de (Gu and Liebe , 2008; Liebe , 2008;
Gu e al., 2010). This model highligh s he lexibili y o he NHEJ
p ocess and explains he di e si y o epai p oduc s gene a ed
om he same ype o DSB. The i e a i e na u e o his p ocess
implies ha mul iple NHEJ componen s can ac on he same
DSB du ing mul iple consecu i e ounds o p ocessing and ha
he in ol emen o ac o s is no mu ually exclusi e o he usage
o o he ones, all o hem emaining ac i e as long as he DSB
con inues un epai ed. On he o he hand, o he au ho s p opose
ha he e is a hie a chy in NHEJ, by which cells gi e p ecedence
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Se ano-Bení ez e al. DNA-End S uc u e and DSB Repai
FIGURE 1 | Unblocking and p ocessing o DSBs. Unblocking pa hways di ec ly con e ends in o 5′-phospah e and 3′-hyd oxyl bu he nucleo ide sequence emains
in ac , p omo ing e o - ee epai (le ). P ocessing can also acili a e blocked DSBs epai emo ing abe an s uc u es om DNA ends by nucleo ide imming
( igh ). This pa hway can lead o e o -p one epai when non- empla ed epai pa hways such as NHEJ o MMEJ a e used. 5′blocks a e depic ed bu simila
si ua ions could be gene a ed on 3′ends.
FIGURE 2 | S uc u e o DNA ends and unblocking enzymes. DSB e mini can be blocked by nume ous chemical s uc u es in i o. Se e al unblocking enzymes a e
p esen in mammalian cells and e icien ly con e hese s uc u es o clean 5′-phospha e and 3′-hyd oxyl DSB e mini (le ). These clean DSBs can be in heo y,
di ec ly epai ed wi h he only enzyma ic ac i i y o a ligase ( igh ).
o esolu ion pa hs wi h he ewes numbe o enzyma ic s eps.
This way, di ec liga ion is a o ed o e mo e complex pa hways
ha include end-p ocessing and/o DNA syn hesis (Wa e s e al.,
2014). Consis en wi h his, LIG4 is he mos lexible ligase known
so a (Ma e al., 2004; Gu e al., 2007), and he di e ences in
how hei ca aly ic domains in e ac wi h di e en end s uc u es
igge d ama ic changes in he dynamics o he en i e NHEJ
complex, de e mining he s eps aken o comple e epai and he
ac o s equi ed (Conlin e al., 2017). A hie a chical o de in he
ac ion o NHEJ componen s is also suppo ed by he o ma ion
o a synapsis wi h wo di e en s ages (G aham e al., 2016). Fi s ,
DNA ends a e e he ed su icien ly a apa , and a e hen closely
aligned by DNA-PK, XLF, and he LIG4-XRCC4 complex. I has
been sugges ed ha his s uc u al con e sion can be coo dina ed
wi h end-p ocessing by changes in he phospho yla ion p o ile
o DNA-PKcs (G aham e al., 2016), which would p o ide a
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Se ano-Bení ez e al. DNA-End S uc u e and DSB Repai
mechanism o he egula ion o end p ocessing and liga ion.
Al hough bo h models could seem con adic o y, hey may no
be mu ually exclusi e. While, NHEJ could beha e as an i e a i e
p ocess in which a ious componen s can be loaded and ac in
a ious combina ions wi hou an es ablished o de , p o iding
lexibili y and e iciency o he epai p ocess, he decision o
how complex ends a e epai ed should no be s ochas ically
de e mined, as speci ic unblocking ac i i ies mus be p e e ed
o e end-p ocessing in o de o a oid sequence modi ica ion.
NUCLEASES IN NHEJ
As men ioned abo e, unde ce ain ci cums ances, DSBs equi e
end p ocessing by he ac ion o nucleases. Usually, hese
nucleases emo e chemical modi ica ions and blockages o
clea e misma ched ends by imming 5′o 3′ e mini h ough
exo- and/o endonucleoly ic p ocessing o expose sho egions
o mic ohomology be ween s ands and p omo e end joining
(Pannunzio e al., 2018). ARTEMIS is he majo nuclease
implica ed in end-p ocessing du ing NHEJ (Ma e al., 2002;
Gooda zi e al., 2006; Yannone e al., 2008). I s main ole
akes place du ing V(D)J ecombina ion, whe e i is esponsible
o he opening o DNA hai pins o med a coding join s, an
endonucleoly ic ac i i y ha is p omo ed by phospho yla ion in
he ABCDE clus e o DNA-PKcs. Howe e , i has been also
shown o ha e DNA-PKcs-independen 5′exonuclease ac i i y on
ssDNA (Pawelczak and Tu chi, 2010; Li e al., 2014). Beyond i s
ole in V(D)J ecombina ion, ARTEMIS con ibu ion in NHEJ
is s ill unde s udy, and ecen analysis demons a ed ha he
ARTEMIS-DNA-PKcs complex also p omo es he liga ion o
incompa ible o e hangs in i o (Chang e al., 2016; Pannunzio
e al., 2018). Besides i s e sa ili y o ac a many di e en ypes o
DNA ends, he e is a common ea u e in all ARTEMIS subs a es:
a ss-dsDNA bounda y, which is p esen in a wide a ie y o
di e en DNA end con igu a ions (Chang e al., 2015; Chang
and Liebe , 2016). In e es ingly, a no el 3′endonuclease ac i i y
o ARTEMIS has been ecen ly desc ibed, ha is p omo ed
by XRCC4-LIG4 complex and also independen o DNA-PKcs
(Ge odimos e al., 2017). The s imula ion o his ac i i y could
be as a esul o a con o ma ional change due o he in e ac ion
wi h LIG4 (Pannunzio e al., 2018).
Ano he ac o in ol ed in he epai o complex ends
equi ing end p ocessing is he MRE11 p o ein om he MRN
complex (consis ing o MRE11, RAD50, and NBS1). The MRN
complex ac s as a senso o DSBs and p omo es epai by NHEJ
o HR. Speci ically, MRE11 exhibi s 3′-5′exonuclease and single-
s anded and DNA hai pin endonuclease ac i i ies (Paull and
Gelle , 1998; T ujillo e al., 2003; Lisby e al., 2004; S acke and
Pe ini, 2011; Williams e al., 2011). Endonucleoly ic clea age
may be o pa icula impo ance o DNA ends co alen ly-
bound o Spo11 (Neale e al., 2005), e mina ed by hai pins
(Lobache e al., 2002) o gene a ed by TOP1 and 2 poisons
(Ha suike e al., 2009; Quenne e al., 2011; Hoa e al., 2016).
Fu he mo e, ecen in i o s udies desc ibed ha NBS1 is
essen ial o p omo e MRE11 nuclease ac i i ies on DNA ends
con aining p o ein adduc s, while i inhibi s MRE11 3′ o 5′
exonuclease deg ada ion o clean ends (Deshpande e al., 2016).
Addi ionally, he unc ion o he MRN complex du ing esec ion
is s imula ed by he phospho yla ed o m o CTIP (Anand
e al., 2016). Rema kably, he nuclease ac i i y o CTIP has been
epo ed o be speci ically equi ed o p ocessing complex DSBs,
such as hose ha bo ing opoisome ase adduc s o gene a ed by
i adia ion. This sugges s ha he endonuclease ac i i y o CTIP
is only necessa y o he emo al o DNA adduc s and no o
he esec ion o unmodi ied DNA b eaks (Makha ash ili e al.,
2014). This di e en ia es ca aly ic and non-ca aly ic unc ions
o CTIP du ing end esec ion, which equi emen would be
end-s uc u e dependen .
POLYMERASES IN NHEJ
As men ioned abo e, as a consequence o he p ocessing o
complex DSBs, he pa icipa ion o o he accesso y ac o s such
as DNA polyme ases o he PolX amily is o en equi ed. These
polyme ases a e especially sui ed o illing in he small gaps ha
a e gene a ed when wo ssDNA p o uding ends wi h he same
pola i y and ha e ei he none o pa ial complemen a i y. The
ac ion o he di e en PolX polyme ases du ing NHEJ seems
o be de e mined by a g adien o empla e s and dependence
a e DSB ends a e synapsed, wi h Polλbeing comple ely
empla e-dependen , Polµha ing some empla e equi emen s
and Te minal Deoxynucleo idyl T ans e ase (TdT) being ully
empla e-independen (McElhinny e al., 2005). The e o e, when
3′-p o uding ends a DSBs do no ha e any complemen a i y
wi h each o he , Polµand TdT polyme ases can add nucleo ides
o gene a ing de no o e minal mic ohomology a DNA ends
(Gu e al., 2007; Da is e al., 2008; Chang e al., 2016).
PolX polyme ases a e ec ui ed o DSBs h ough he speci ic
in e ac ion be ween hei BRCT domains wi h NHEJ co e ac o s
(Muelle e al., 2008; Boubakou -Azzouz e al., 2012; Malu e al.,
2012; C ax on e al., 2018). These in e ac ions a o DSB epai
e iciency (Tseng and Tomkinson, 2002; C ax on e al., 2018),
and can be acili a ed o some ex en by DNA-PKcs-media ed
phospho yla ion (Sas e-Mo eno e al., 2017). In ac , sys ema ic
analyses o de e mine how o e hang sequence a ec s he ac i i y
o NHEJ polyme ases has shown some DNA syn hesis pa e ns
ha may be coo dina ed wi h liga ion complex capabili ies
(C ax on e al., 2018).
END-PROTECTING FACTORS
In addi ion o all hese unblocking and p ocessing ac o s, o he
accesso y NHEJ componen s a e equi ed o inhibi o es ic
deg ada ion o DSB ends, and he e o e a oid excessi e DNA
sequence loss. In his ega d, modi ica ions a he ch oma in
lanking he DSB, such as his one H2AX phospho yla ion
(Helmink e al., 2011), and he subsequen ec ui men o
downs eam ac o s o he DNA damage esponse (DDR), such as
MDC1, 53BP1, and BRCA1 (Bekke -Jensen and Mailand, 2010)
ep esen c ucial e en s o he choice o p ope epai pa hways,
egula ing o which ex en DSB ends a e p ocessed. Acco dingly,
H2AX de icien mice show an inc ease in genome ins abili y and,
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Se ano-Bení ez e al. DNA-End S uc u e and DSB Repai
in he absence o P53, a e p one o umo de elopmen (Celes e
e al., 2002, 2003; Bassing e al., 2003). Mo eo e , in ARTEMIS
de icien cells, H2AX was epo ed o limi he p ocessing o
DNA ends by CTIP endonuclease upon induc ion o blocked
DSBs du ing V(D)J ecombina ion, his unc ion o H2AX being
media ed by MDC1 (Helmink e al., 2011). In he same way,
53BP1 has been also shown o egula e end-p ocessing du ing
V(D)J and CSR ecombina ion (Di ilippan onio e al., 2008;
Bo hme e al., 2010) and o inhibi CTIP-dependen esec ion
in BRCA1 de icien cells a pos - eplica i e s ages o cell cycle,
sugges ing ha H2AX phospho yla ion may es ic esec ion by
he ec ui men o 53BP1 (Bun ing e al., 2010). The p o ec i e
ole o DNA ends by 53BP1 equi es he pa icipa ion o some
downs eam ac o s, such as PTIP (Ku imasa e al., 2015) and
RIF1 (Kienke , 2000; Lee K. J. e al., 2004; Douglas e al., 2005),
and maybe o he ac o s ye o be disco e ed. In his ega d,
he ecen ly disco e ed ssDNA-binding complex shieldin has
been p oposed o ac as ul ima e e ec o o he 53BP-RIF1
pa hway o end p o ec ion (Chan e al., 2002; Ding e al.,
2003; Meek e al., 2007). O no e, ARTEMIS was p e iously
iden i ied as a PTIP-binding p o ein, and, s ikingly, as one o
main downs eam e ec o s o 53BP1-PTIP pa hway (Wang e al.,
2014). This sugges s ha 53BP1 could be p omo ing limi ed end-
imming and he epai o DSBs h ough NHEJ, and he e o e
di ec ly compe ing wi h he HR epai pa hway ha would en ail
long esec ion.
DNA-PKcs, A MASTER REGULATOR OF
ACCESS TO DSB ENDS
Despi e no being conse ed in lowe euka yo es, he ac i i y o
his phospha idylinosy ol 3-kinase- ela ed kinase (PI3KK) is a
clea equisi e o i s unc ioning du ing NHEJ in mammalian
cells (Kienke , 2000; Ku imasa e al., 2015). Al hough he e is
a long lis o DNA-PKcs subs a es, mu a ional analysis (Lee K.
J. e al., 2004; Douglas e al., 2005; Gooda zi e al., 2006; Meek
e al., 2008) concludes ha DNA-PKcs i sel is he only NHEJ
ac o ha has been shown o be a unc ionally ele an a ge
o i s own kinase ac i i y (Chan e al., 2002; Ding e al., 2003;
Soubey and e al., 2003; Cui e al., 2005; Douglas e al., 2007;
Meek e al., 2007, 2008). The mos well-accep ed consequence
o such DNA-PKcs au ophospho yla ion is i s inac i a ion and
dissocia ion om DNA ends, allowing subsequen joining by
LIG4 (Chan and Lees-Mille , 1996; Douglas e al., 2001). Despi e
he ac ha DNA end binding by DNA-PKcs is indi e en o
dis inc DNA end s uc u es, some s udies indica e ha cispla in-
DNA adduc s nea he ends educe kinase ac i a ion, sugges ing
ha ee e mini could be in ol ed in he ac i a ion o DNA-PKcs
(Tu chi, 2000; Pawelczak e al., 2005). I has been sugges ed ha
kinase ac i a ion occu s in ans, linking au ophospho yla ion
o DNA-PKcs o synapsis. Al hough his poin is s ill a ma e
o deba e, his may p o ide an impo an mechanism by which
DNA-PKcs p o ec s DNA-ends o main ain genomic in eg i y.
Howe e , ex ensi e s udies ha e shown ha in esponse o DSBs,
DNA-PKcs au ophospho yla ion can occu in di e en esidues,
wi h each e en ha ing speci ic unc ional consequences (Meek
e al., 2008; Da is e al., 2014). In human DNA-PKcs, amino acid
clus e s known as ABCDE, lanking Th 2609 esidue, and PQR,
a ound he Se 2056 esidue, a e he wo majo phospho yla ion
si es (Ding e al., 2003; Block e al., 2004; Reddy e al., 2004;
Cui e al., 2005; Meek e al., 2007). Al hough bo h clus e s can
be au ophospho yla ed by DNA-PKcs i sel , he ABCDE clus e
can be also phospho yla ed by ATM o ATR unde di e en
cellula s esses (Chen e al., 2007; Meek e al., 2008; Da is e al.,
2010). Si e-di ec ed mu agenesis analyses and cha ac e iza ion
o animal models o DNA-PKcs de iciency (Blun e al., 1996;
A aki e al., 1997; Taccioli e al., 1998; Beamish e al., 2000;
Zhang e al., 2011; Danska e al., 2015; Jiang e al., 2015)
ha e e ealed ha he speci ic de ec esul ing om blocking
ei he ABCDE o PQR phospho yla ion is DNA end p ocessing
de egula ion. Bo h clus e s show an agonis ic unc ions, and
whe eas phospho yla ion in he ABCDE clus e p omo es DNA
end p ocessing, phospho yla ion o si es wi hin he PQR clus e
inhibi s DNA end esec ion. Speci ically, he ABCDE clus e is
epo ed o p omo e end p ocessing by egula ing he access
o ARTEMIS o he ends (Ma e al., 2002; Cui e al., 2005;
Gooda zi e al., 2006; Yannone e al., 2008). On he o he hand,
end-liga ion equi es a s ic DNA-PKcs au ophospho yla ion,
possibly in he PQR clus e , which is p omo ed by liga able ends
and synapsis. This way, possible unsuccess ul liga ion a emp s
a e a oided. Thus, DNA-PKcs can be conside ed a molecula
shi ha coo dina es end p ocessing and liga ion h ough i s
phospho yla ion o maximize he e iciency o he NHEJ pa hway.
ATM, A KEY FACTOR TO ORCHESTRATE
END PROCESSING
A axia Telangiec asia Mu a ed (ATM) kinase is ano he membe
o he PI3KK amily, ecognized by i s unc ion as an apical
ac i a o o he DDR in esponse o DSBs (McKinnon,
2004). In e es ingly, he s uc u e o ends is a c ucial ac o
which de e mines he equi emen o ATM o he epai
o a DSB (Ál a ez-Quilón e al., 2014). Speci ically, ATM
exclusi ely acili a es he epai o i e e sibly blocked TOP2-
media ed DSBs, a ising by e oposide ea men in TDP2-
de icien backg ound (Ál a ez-Quilón e al., 2014). Consis en
wi h his, ATM-media ed epai p omo es cell su i al and he
main enance o genome in eg i y, a oiding mic onuclei and
ch omosomal abe a ion o ma ion a e he induc ion o DSBs
ha bo ing e mini ha equi e end p ocessing (Ál a ez-Quilón
e al., 2014). Al hough he unde lying molecula mechanisms
by which ATM deals wi h blocked DNA ends a e s ill unclea ,
wo complemen a y explana ions ha e been p oposed (Ál a ez-
Quilón e al., 2014). On he one hand, ATM can p omo e
limi ed esec ion o elimina e he complex s uc u es a DSB
ends h ough he ac ion o nucleases. In his ega d, ATM
phospho yla es ARTEMIS and DNA-PKcs a he ABCDE clus e
(see abo e) (Chen e al., 2007; Meek e al., 2008; Da is e al.,
2010). In addi ion, a unc ional in e play be ween ATM and
he MRN complex has been widely epo ed. Indeed, he h ee
componen s o he complex a e all phospho yla ed by ATM,
which has been p oposed as a modula o o i s p ocessing ac i i y
F on ie s in Molecula Biosciences | www. on ie sin.o g 5Janua y 2020 | Volume 6 | A icle 153

Se ano-Bení ez e al. DNA-End S uc u e and DSB Repai
(Kijas e al., 2015). Then, he MRN complex in e ac s wi h C IP,
which is also posi i ely egula ed by ATM o p omo e end-
esec ion (You and Bailis, 2010; Wang e al., 2013). Finally, ATM
egula es o he nucleases ha could be in ol ed in esol ing
incompa ible ends. This includes APLF (Ap a axin and PNKP-
like ac o ) (Mac ae e al., 2008; Fen on e al., 2013); DNA
eplica ion helicase/nuclease 2 (DNA2) (Paudyal e al., 2017) o
EXO1 (Bolde son e al., 2010; Tomima su e al., 2017). On he
o he hand, ATM could es ic excessi e nucleoly ic deg ada ion
o DNA ends (Rahal e al., 2008). This can ac ually ope a e
by a di ec inhibi o y ac ion on a o emen ioned nucleases such
as MRE11 (Rahal e al., 2010) o EXO1 (Bolde son e al.,
2010), and/o by p omo ing modi ica ions a he ch oma in
lanking he DSB and he ec ui men o p o ec ing ac o s.
In his ega d, he p o ec i e unc ion o H2AX depends on
i s phospho yla ion a Se 139 o o m γ-H2AX in ch oma in
lanking DNA DSBs (Helmink e al., 2011), which is p e e en ially
ca ied ou by ATM (Takahashi e al., 2010). The γ-H2AX
downs eam ac o MDC1 is also phospho yla ed by ATM,
p omo ing i s oligome iza ion and sp eading on ch oma in
(Ma échal and Zou, 2013). In addi ion, ATM phospho yla es
53BP1 (Ande son e al., 2002; Jowsey e al., 2007) and hese
phospho yla ions a e equi ed o 53BP1 in e ac ion wi h PTIP
(Munoz e al., 2007) and RIF1 (Chapman e al., 2013). Finally,
in addi ion o hese dual end p ocessing/-p o ec i e oles, ATM
could ope a e a a la e s age in he epai p ocess. Fo example,
a e ionizing adia ion-induced DSBs, ATM phospho yla es
Polλ, which would p omo e con o ma ional changes in Polλ
ha acili a e i s in e ac ion wi h NHEJ co e ac o s a
DSBs and, hence, s imula es gap- illing DNA syn hesis du ing
NHEJ (Sas e-Mo eno e al., 2017).
The s uc u e and con o ma ion o DNA ends a e he e o e
de e minan o he epai p ocess and ou come, especially
in si ua ions in which end-joining mechanisms a e p e alen .
Al hough many o he enzyma ic ac i i ies equi ed ha e been
iden i ied and cha ac e ized in de ail, he mechanisms by which
cells egula e and in eg a e hese ac i i ies o keep sequence
a ia ion unde con ol a e s ill poo ly unde s ood. In his sense,
i is emp ing o hink on blocked DSBs and a de egula ed
cellula esponse o hese lesions as impo an h ea s o genome
in eg i y, and, po en ially, d i e s o malignan ans o ma ion
and cance .
AUTHOR CONTRIBUTIONS
AS-B, FC-L, and JR concei ed and w o e he manusc ip .
ACKNOWLEDGMENTS
Wo k in he FC-L labo a o y was unded wi h g an s om
he Spanish and Andalusian Go e nmen (SAF2017-89619-R,
CVI-7948, Eu opean Regional De elopmen Fund), and he
Eu opean Resea ch Council (ERC-CoG-2014-647359); and wi h
an indi idual ellowship o AS-B (Beca P edoc o al AEFAT,
Asociación Española Familia A axia Telangiec asia). CABIMER
was suppo ed by he Andalusian Go e nmen .
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