DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein

Published online 9 May 2017 Nucleic Acids Resea ch, 2017, Vol. 45, No. 12 7237–7248 doi: 10.1093/na /gkx395 DNA syn hesis de e mines he binding mode o he human mi ochond ial single-s anded DNA-binding p o ein Jos´ eA.Mo in 1,†, Fe nando Ce ´ on1,†, Ja ie Ja illo2,†, Elena Bel an-He edia2, G zego z L. Ciesielski3,4, J. Rica do A ias-Gonzalez1,5, Lau ie S. Kaguni3,4,*, F ancisco J. Cao2,* and Bo ja Iba a1,5,* 1Ins i u o Mad ile˜ no de Es udios A anzados en Nanociencia, IMDEA Nanociencia, 28049 Mad id, Spain, 2Depa amen o F´ ısica A ´ omica, Molecula y Nuclea , Uni e sidad Complu ense, 28040 Mad id, Spain, 3Ins i u e o Biosciences and Medical Technology, Uni e si y o Tampe e, 33520 Tampe e, Finland, 4Depa men o Biochemis y and Molecula Biology and Cen e o Mi ochond ial Science and Medicine, Michigan S a e Uni e si y, Eas Lansing, MI 48823, USA and 5Ins i u o Mad ile˜ no de Es udios A anzados en Nanociencia (IMDEA Nanociencia) and CNB-CSIC-IMDEA Nanociencia Associa ed Uni ‘Unidad de Nanobio ecnolog´ ıa’, 28049 Mad id, Spain Recei ed Decembe 21, 2016; Re ised Ap il 19, 2017; Edi o ial Decision Ap il 25, 2017; Accep ed Ap il 27, 2017 ABSTRACT Single-s anded DNA-binding p o eins (SSBs) play a key ole in genome main enance, binding and o ga- nizing single-s anded DNA (ssDNA) in e media es. Mul ime ic SSBs, such as he human mi ochond ial SSB (Hm SSB), p esen mul iple si es o in e ac wi h ssDNA, which has been shown in i o o enable hem o bind a a iable numbe o single-s anded nucleo ides depending on he sal and p o ein con- cen a ion. I has long been sugges ed ha di e en binding modes migh be used selec i ely o di e - en unc ions. To s udy his possibili y, we used op- ical weeze s o de e mine and compa e he s uc- u e and ene ge ics o long, indi idual Hm SSB–DNA complexes assembled on p e o med ssDNA and on ssDNA gene a ed g adually du ing ‘ in si u ’ DNA syn- hesis. We show ha Hm SSB binds o p e o med ss- DNA in wo majo modes, depending on sal and p o- ein concen a ion. Howe e , when p o ein binding was coupled o s and-displacemen DNA syn hesis, only one o he wo binding modes was obse ed un- de all expe imen al condi ions. Ou esul s e eal a key ole o he g adual gene a ion o ssDNA in mod- ula ing he binding mode o a mul ime ic SSB p o- ein and consequen ly, in gene a ing he app op ia e nucleop o ein s uc u e o DNA syn he ic eac ions equi ed o genome main enance. INTRODUCTION Single-s anded DNA-binding p o eins (SSBs) a e com- ponen s o e e y nucleic acid ansac ion ha equi es single-s anded in e media es. They bind p e e en ially o single-s anded DNA (ssDNA) wi h high a ini y and in a sequence independen manne , p o ec ing ssDNA om deg ada ion and a he same ime, de ining he nucleop o- ein subs a e upon which o he p o eins mus ac (1). Mo e- o e , SSB p o eins in e ac physically wi h a b oad a ay o genome main enance p o eins, a ge ing hem o hei si es o ac i i y and s imula ing hei biochemical unc ions (2). The as majo i y o SSB p o eins p esen mul iple si es o in e ac wi h ssDNA (i.e. OB olds) (3,4), which enable hem o in e ac in i o wi h a a iable numbe o nucleo ides de- pending on he expe imen al condi ions (2). As shown o he Esche ichia coli SSB (EcoSSB), hese binding modes a e e e sibly in e con e ible depending on he sal concen a- ion and ype, as well as p o ein binding densi y on he ss- DNA (5–7). Mi ochond ial SSB (m SSB) is a hallma k o eplica ing mi ochond ial nucleoids; i is equi ed o main ain he copy numbe o m DNA (8–10) and plays c ucial oles du ing he ini ia ion (11,12) and elonga ion phases o mi ochon- d ial DNA eplica ion, co e ing he pa en al hea y s and and in e ac ing unc ionally wi h he o he componen s a he eplica ion o k (13–17). In addi ion, m SSB has been *To whom co espondence should be add essed. Tel: +34 91 2998863; Fax: +34 91 2998730; Email: bo ja.iba [email p o ec ed] Co espondence may also be add essed o Lau ie S. Kaguni. Tel: +517 3536703; Fax: 517 3551602; Email: [email p o ec ed] Co espondence may also be add essed o F ancisco J. Cao. Tel: +34 91 3944742; Fax: +34 913945193; Email: [email p o ec ed] †These au ho s con ibu ed equally o he pape as i s au ho s. P esen add ess: Jos´ e A. Mo in, Bio echnology Cen e (BIOTEC). Technical Uni e si y D esden. Ta zbe g 47/49, 01307 D esden, Ge many. C The Au ho (s) 2017. Published by Ox o d Uni e si y P ess on behal o Nucleic Acids Resea ch. 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 (h p://c ea i ecommons.o g/licenses/by-nc/4.0/), which pe mi s non-comme cial e-use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Fo comme cial e-use, please con ac jou nals.pe [email protected] 7238 Nucleic Acids Resea ch, 2017, Vol. 45, No. 12 shown o acili a e packaging o m DNA in o nucleoid-like s uc u es (18), modula e he le el m DNA ansc ip ion (19) and may be in ol ed in he p ocess o DNA epai (20). Thus, m SSB is essen ial o mi ochond ial unc ion and in u n, o cell iabili y. Human mi ochond ial SSB (Hm SSB) binds ssDNA as a e ame composed o ou iden ical 16 kDa subuni s, wi h each subuni con aining an OB- old (21). The o e all olding o he Hm SSB e ame esembles ha o EcoSSB, wi h which i sha es a numbe o conse ed esidues wi hin he amino- e minal egion (22). Fluo escence i a ion s udy on polydT o Hm SSB showed appa en binding-si e sizes o 50–70 n pe e ame be ween 50 mM and 2 M NaCl (23), sugges ing ha he human p o- ein may unde go sal -dependen binding mode ansi ions simila o hose obse ed wi h EcoSSB. I has been sug- ges ed ha because he p ope ies o hese w apping modes di e signi ican ly, hey may be used p e e en ially in a - ious DNA me abolic p ocesses (2,7,24,25). Al hough indi- ec suppo o his p oposal has been published (26–31), o da e he e is no di ec expe imen al e idence o suppo which i any o hese binding modes is selec ed p e e en ially when binding is coupled o he g adual gene a ion o ss- DNA du ing a DNA me abolic p ocess, such as DNA epli- ca ion. Single-molecule o ce spec oscopy s udies ha e p o en use ul o in es iga e he si e size, equilib ium cons an s and ene ge ics o he binding o a ious SSB p o eins o ssDNA (32–37). He e, in o de o de e mine he ole o DNA syn- hesis in speci ying he mode o Hm SSB binding o ss- DNA, we used op ical weeze s o measu e and compa e he elas ic and ene ge ic p ope ies o long nucleop o ein complexes, ei he assembled on p e o med ssDNA o as- sembled ‘in si u’ du ing syn hesis a a dsDNA o k. The dy- namics o long nucleop o ein–DNA complexes is especially ele an when s udying Hm SSB, because long ssDNA in- e media es may accumula e ansien ly du ing eplica ion o he mi ochond ial genome (38). Modeling o he o ce ex ension cu es (FECs) o indi idual nucleop o ein com- plexes e ealed he p o ein-binding mode, i.e. he a e age numbe o nucleo ides w apped pe e ame : when assem- bled on p e o med ssDNA molecules, Hm SSB can bind o he ssDNA in wo majo modes, which depend on sal and p o ein concen a ion. Howe e , when p o ein binding was coupled o DNA syn hesis, only one o he wo binding modes was obse ed unde all expe imen al condi ions. Ou esul s e eal a key ole o he g adual elease o ssDNA du ing DNA syn hesis in egula ing he Hm SSB binding mode and consequen ly, on gene a ing he app op ia e nu- cleop o ein s uc u e o subsequen eplica ion o he dis- placed s and. Simila ly, he p og essi e elease o ssDNA du ing o he DNA me abolic p ocesses is expec ed o egu- la e he binding modes o p oka yo ic and euka yo ic SSB p o eins ha expose mul iple OB olds. MATERIALS AND METHODS P o eins and DNA cons uc s Recombinan Hm SSB p o ein was p epa ed om bac e ial cells as desc ibed p e iously (39,40). Bac e iophage Phi29 DNA polyme ase was pu i ied as desc ibed elsewhe e (41). The DNA hai pins we e p epa ed as ollows. The main unwinding s em o he hai pin was syn hesized by polyme ase chain eac ion ampli ica ion o a 2500 bp DNA segmen o he bac e iophage Phi29 genome and subsequen ly diges ed wi h EcoRI and SalI e- s ic ion endonucleases. The SalI end was liga ed o a sel -annealing oligonucleo ide o ming a pen a-loop (5- TCGAGCAGATGCAGCAATAACGTGCATCTGC-3). The EcoRI end was liga ed o a 30 bp dsDNA linke labeled wi h bio in a he 5-end and wi h digoxigenin a he 3-end. The inal leng h o he hai pin cons uc is 2540 bp. Digoxigenin labeling was desc ibed elsewhe e (42,43). To measu e p o ein binding and he co esponding FECs a 300 mM NaCl and 50 mM NaCl/4mMMgCl 2, he hai - pin was modi ied o include a 2686 bp DNA handle (pUC19 ec o , No agen, diges ed wi h Ps I and BamHI es ic ion endonucleases) be ween he linke and digoxigenin label. To de e mine he binding o Hm SSB du ing DNA syn hesis, he la e hai pin was modi ied u he o include an abasic si e a he pen a-loop ha o ms he end o he hai pin. Op ical weeze s expe imen s We used a coun e p opaga ing dual-beam op ical weeze s ins umen (44) o manipula e indi idual DNA hai pins e he ed be ween an op ically apped an i-digoxigenin- coa ed bead and a s ep a idin-coa ed bead immobilized on op o a mic opipe e. To measu e Hm SSB binding o p e o med ssDNA, he DNA hai pin was mechanically opened by g adually applying inc easing o ce o he ends o he complemen a y s ands. Unwinding o he hai pin was pe o med in he p esence o 250 nM oligonucleo ide complemen a y o he egion a he apex o he hai pin (5- GCCGATGCACGTTATTCGTGCATCGGCTCG-3). Once he hai pin was opened (F ∼19 pN), hyb idiza ion o he oligonucleo ide a he apex gene a ed a la ge kine ic ba ie o hai pin e-annealing when ension is eleased, p o iding a me hod o measu e he o ce-ex ension cu e o he esul ing 5080 ssDNA (45). The oligonucleo ide binds speci ically o he apex o he hai pin and con ibu es ≤0.6% o he inal leng h o he molecule. The e o e, we conside ed i has no signi ican e ec on he inal ex ension o he FECs. A e washing ou he oligonucleo ide, he Hm SSB p o ein was in oduced inside he low cell a e dilu ion o 5, 10, 50, 100 o 200 nM in he app op ia e eac ion bu e . The eac ion bu e s con ained 50 mM T is–HCl, pH 7.5, 2 mM Di hio h ei ol (DTT) and 10, 50, 100 o 300 mM o NaCl and 4 mM MgCl2when indica ed. To de e mine he DNA unw apping and p o ein de ach- men o ces, he nucleop o ein complexes we e assembled as desc ibed abo e using 10 o 100 nM o Hm SSB dilu ed in eac ion bu e s con aining 10, 50 o 300 mM o NaCl, 50 mM NaCl plus 4 mM MgCl2o 300 mM NaCl plus 4 mM MgCl2. To de e mine he binding o Hm SSB du ing DNA syn- hesis, eac ions we e pe o med in bu e s con aining 50 mM T is–HCl, pH 7.5, 2 mM DTT, 50 mM NaCl, 4 mM MgCl2,50␮M dNTPs, 2 nM Phi29 polyme ase in he p es- ence o 5, 50, 100 o 200 nM Hm SSB. Bo h, Hm SSB binding o p e o med ssDNA and o ssDNA g adually gene a ed du ing DNA syn hesis we e moni o ed a 60 Hz a 22 ±1◦C in he ‘cons an o ce eed- Nucleic Acids Resea ch, 2017, Vol. 45, No. 12 7239 back’ mode, in which he dis ance be ween he beads was adjus ed o main ain a cons an ension in he DNA (F =3 ±1 pN). In all cases, a e comple ion o he binding eac- ion, he excess o Hm SSB was washed ou om he cham- be wi h he co esponding eac ion bu e . Finally, he FEC o he Hm SSB–DNA complex was ob ained by pulling he complex a a a e o 200 nm/s. O e all we es ed o e 30 di e en expe imen al condi ions and collec ed 5–10 FECs pe condi ion. Da a analysis The FECs o ba e ssDNA we e i wi h he eely-join ed- chain model (FJC) and he ex ensible wo m-like-chain models (XWLC) o polyme elas ici y (46)(Figu e1and Supplemen a y Figu e S1). The amoun o seconda y s uc- u e o ma ion on he ssDNA as a unc ion o mechanical ension a each sal ype and concen a ion was calcula ed acco ding o Bosco e al.(45) (Supplemen a y Figu e S2). The ex ension pe nucleo ide o he ee ssDNA, XssDNA(F) in Equa ion (1), was ob ained di ec ly om he expe imen- al FECs o he ssDNA a each sal ype and concen a- ion. The o al ex ension o hyb id molecules con aining ds- DNA and ssDNA segmen s is gi en by he sum o he ex en- sions o hei dsDNA and ssDNA po ions a each ension (33,42) (Supplemen a y Figu e S3). Calcula ion o he Hm SSB-ssDNA w apping ee ene gy. The a e age wo k o unw ap 1 n om each e ame , ΔGw, in pN·nm, was con e ed o kBTuni s (Bol zmann cons an mul iplied by he absolu e empe a u e) using he ela ion- ship kBT=4.11 pN·nm a 25◦C, which co esponds o he ene gy con ibu ion om he mal luc ua ions. Calcula ion o he DNA syn hesis a e. The numbe o nu- cleo ides inco po a ed by he Phi29 DNA polyme ase as a unc ion o ime was ob ained by di iding he obse ed dis ance change be ween he beads by he a e age change in ex ension a a gi en ension accompanying he gene a- ion o one new base pai and ei he one ee nucleo ide (in he absence o Hm SSB) o one Hm SSB-bound nucleo ide (43) (Supplemen a y Figu e S8). The a e age a es we e de- e mined by a line i o he aces showing he numbe o eplica ed nucleo ides e sus ime. RESULTS Fo ce ex ension cu es o Hm SSB–DNA complexes assem- bled on p e o med ssDNA molecules a y wi h solu ion con- di ions We i s s udied he mechanical p ope ies o indi idual p o ein–DNA complexes assembled on p e o med ssDNA molecules. We used he op ical weeze s o unwind a sin- gle DNA hai pin o 2540 bp in he p esence o a 30-me oligonucleo ide complemen a y o he loop egion o he hai pin, Figu e 1A (see ’Ma e ials and Me hods’ sec ion). Once he hai pin is ully unwound (F ∼19 pN), he speci ic annealing o he oligonucleo ide o he loop egion p e en s he e-winding o he hai pin as ension is eleased, p o id- ing a me hod o measu e he FEC o he esul ing 5080-n long ssDNA (Supplemen a y Figu e S1) (45). A e wash- ing ou he oligonucleo ide, Hm SSB dilu ed in an app o- p ia e eac ion bu e was passed inside he low cell (see be- low). We moni o ed a low cons an o ces (∼3±1 pN) he end- o-end dis ance change o he e he ed ssDNA as he Hm SSB p o ein bound o and o ganized he ssDNA (x, Figu e 1B). This low o ce ange was chosen o minimize he po en ial e ec o mechanical ension on he w apping s a e o he ssDNA a ound he Hm SSB e ame s (33). Once equilib ium was eached (when no u he dis ance changes we e obse ed, Figu e 1B), excess Hm SSB was washed ou o he low cell (so ha no p o ein ebinding is hen ex- pec ed) and ension was ini ially dec eased o ze o and hen inc eased o 30–40 pN, in o de o ob ain he inal FEC o he Hm SSB–DNA complex (Figu e 1C). Because he Hm SSB binding mode may depend on sal and p o ein concen a ion (23,39), he nucleop o ein com- plexes we e assembled in eac ion bu e s con aining a i- able le els o NaCl (10, 50, 100 and 300 mM), NaCl and MgCl2(50 and 4 mM, espec i ely) and Hm SSB concen- a ions (5, 10, 50, 100 and 200 nM). We hen es ed ∼25 di e en combina ions, each p esen ing a speci ic a io o Hm SSB and sal concen a ion ([SSB]/[NaCl]), which is a measu emen o he numbe o p o ein e ame s pe ion in solu ion. Fo all expe imen al condi ions, he s e ch- ing FECs o he Hm SSB–ssDNA complexes displayed an en i ely di e en ex ension as compa ed o he absence o p o ein, esul ing om he o ganiza ion o he ssDNA by Hm SSB. Thei o e all shape depends on he sal concen a- ion and ype and on Hm SSB concen a ion (Figu e 1C). As desc ibed p e iously in bulk s udies, we did no obse e any e idence suppo ing Hm SSB agg ega ion unde ou cu en expe imen al condi ions (39,47). We no e ha o condi ions ha sho ened signi ican ly he end- o-end dis ance o he ssDNA a low ension (300 mM NaCl o 4 mM MgCl2, Figu e 1C and Supplemen a y Figu e S2), we included a long dsDNA handle o ∼1␮m on he 3-end o he hai pin (see ’Ma e ials and Me hods’ sec ion) o p e en non-speci ic p o ein–DNA-bead in e - ac ions ha migh in e e e wi h he de e mina ion o he FECs. The dsDNA handle does no a ec he elas ic p op- e ies o he ssDNA (33,42) (Supplemen a y Figu e S3). A he highes p o ein concen a ion used in his wo k (200 nM), mammalian m SSBs p o eins do no bind dsDNA (48). Fo ces p omo ing o e all DNA unw apping and p o ein de- achmen E alua ion o he in insic elas ic p ope ies o he Hm SSB–ssDNA complexes is equi ed o de e mine he Hm SSB–ssDNA binding mode unde each expe imen al condi ion. Al hough con ained wi hin he FECs, his in- o ma ion is combined wi h DNA unw apping and p o ein de achmen e en s ha ake place as ension is inc eased (33,34). To de e mine he o ces p omo ing DNA unw ap- ping (Fu) and p o ein de achmen (Fd), and o iden i y he egion o he FECs ha e lec he in insic elas ic p op- e ies o he complex, we pe o med he ollowing expe - imen s. Indi idual p o ein–DNA complexes, assembled as desc ibed abo e unde a ying sal and p o ein concen a- 7240 Nucleic Acids Resea ch, 2017, Vol. 45, No. 12 Figu e 1. Binding o Hm SSB o p e o med ssDNA molecules. (A) Schema ic o he expe imen al se up. The complemen a y s ands o a DNA hai pin (2540 bp) we e e he ed be ween an op ically apped bead (g ay) and a bead on op o a mic opipe e (black) (Ma e ials and Me hods). Mechanical unwinding o he DNA hai pin in he p esence o a 30-me oligonucleo ide (Oligon , ed line) complemen a y o he loop end p e en s e-winding o he hai pin a low o ces, p o iding a me hod o manipula e long ssDNA molecules. A cons an o ce (∼3 pN), w apping o ssDNA by Hm SSB dec eases he dis ance be ween he beads (x). The inal p oduc o he eac ion is a 5080-n long ssDNA–Hm SSB complex. Al e na i ely, a DNA hai pin con aining a dsDNA handle a he 3end was used o measu e he FECs a 300 mM NaCl and 50 mM NaCl/4mMMgCl 2(Ma e ials and Me hods). (B) Independen aces showing he change in ex ension (xinnm/n ) wi h ime due o p o ein binding o he ssDNA (50 nM Hm SSB and 50 mM NaCl). T aces we e shi ed along he ime axis o cla i y o display. (C) Expe imen al FECs o p o ein–DNA complexes assembled unde a ying p o ein concen a ions, sal concen a ions and ype. Fo all panels ed and blue cu es co espond o he s e ching FECs o p o ein–DNA complexes assembled a he indica ed p o ein concen a ions (nM). Two FECs pe p o ein concen a ion condi ion a e shown. Ligh ed and ligh blue cu es co espond o he ini ial ssDNA FECs unde each condi ion. Dashed black line co esponds o he WLC model o ssDNA elas ici y wi h a pe sis ence leng h o 0.75 nm and s e ch modulus o 800 pN. ions (see ’Ma e ial and Me hods’ sec ion), we e subjec ed o a se ies o s e ch– elax cycles in which o ce was in- c eased by 1–2 pN in each consecu i e cycle (Supplemen- a y Figu e S4). Unde all expe imen al condi ions, he s e ching and e- laxing FECs we e e e sible o ensions below 5 ±1pN (Figu e 2A, le cu e); abo e hese o ce alues hys e e- sis e en s we e obse ed. The minimum o ce igge ing he o e all unw apping (Fu) was de e mined as he a e age o ce alue abo e which he s e ch– elax cycle p esen ed hys e esis, bu consecu i e s e ching cycles exhibi ed he same, o iginal ex ensions (Figu e 2A, cen al cu e). Be- cause hese expe imen s we e pe o med in he absence o ee p o ein in solu ion, he eco e y o he ini ial s e ching cycle indica es he e is no ne p o ein de achmen , whe eas he p esence o hys e esis indica es ha o e all unw apping is signi ican a ensions a ound Fu. The minimum o ce ha begins o p omo e p o ein de achmen (Fd) was de e mined as he a e age o ce alue abo e which consecu i e s e ch- ing cycles show di e en ex ensions. When o ce is inc eased abo e Fd, he second s e ching cycle is longe han he i s , indica ing ha se e al Hm SSB e ame s ha e de ached om he ssDNA (Figu e 2A, igh cu e). Analysis o he s e ch– elax cycles showed ha bo h Fuand Fdinc ease as he [SSB]/[NaCl] a io inc eases (Figu e 2B and Supplemen a y Figu e S4). A i o he da a wi h a wo-s a e unc ion indica es ha a change in he [SSB]/[NaCl] a io om ∼0.5·106 o 1·106inc eased ab up ly he alues o Fuand Fd(Figu e 2B). A low p o ein concen a ions and/o high NaCl concen a ions (lowes [SSB]/[NaCl] a ios) o e all unw apping and p o- ein de achmen begin a o ce alues o Fu∼6pNand Fd∼8 pN, espec i ely, whe eas as p o ein concen a ion inc eases and/o NaCl concen a ion dec eases (highes [SSB]/[NaCl] a ios), unw apping and de achmen e en s a e sepa a ed by a wide o ce ange, Fu∼9pNandFd∼14 pN, espec i ely. In e es ingly, he alues ob ained o Fu and Fdag ee well wi h he a e age o ces epo ed p e i- ously o unw ap and de ach a single EcoSSB e ame om ssDNA, 3–8 pN and ∼10 pN, espec i ely (33,34). These simila i ies sugges s ongly ha o bo h SSBs mechanical ension has a e y simila e ec on he o e all o ganiza ion o he DNA a ound he p o ein e ame . We no e ha o all expe imen al condi ions s e ching o ces abo e 30–40 pN a e equi ed o de ach all Hm SSB e ame s om he ssDNA (Supplemen a y Figu e S4). Nucleic Acids Resea ch, 2017, Vol. 45, No. 12 7241 Figu e 2. De e mina ion o DNA unw apping (Fu) and p o ein de ach- men (Fd) o ces. (A) Rep esen a i e (no sequen ial) s e ch– elax cycles o a Hm SSB–DNA complex assembled a 50 mM NaCl/100 nM Hm SSB. Fo all plo s, a e ini ial s e ching ( ed cu e o P1) he o ce was elaxed back o ∼0 pN (black cu e o R) and hen was inc eased again (blue cu e o P2) o a o ce 1–2 pN abo e he ini ial s e ching o ce. Unde all ex- pe imen al condi ions, he FECs exhibi a e e sible egion (P1=R=P2). Abo e a c i ical o ce (see below), hys e esis be ween he s e ching and elaxing cu es was obse ed (highligh ed in g een and pu ple). Hys e e- sis is indica i e o DNA unw apping when he s e ching cu es ha e he same ex ension (P1=P2= R) and o p o ein de achmen when he sequen- ial s e ching cu es show di e en ex ensions (P1= P2= R). Schema ic o he expec ed e en s along he pulling axis (F) is shown on op. Hm SSB is ep esen ed as a b own e ame . (B) Dependencies o Fu(open blue sym- bols) and Fd(black symbols) on he [SSB]/[NaCl] a io. Red symbols ep- esen da a ob ained unde eac ion condi ions con aining 50 mM NaCl plus 4 mM MgCl2.Fuand Fdwe e ob ained om he analysis o he a eas o hys e esis (Supplemen a y Figu e S4). E o ba s ep esen s anda d e - o (s.e.). Solid lines ep esen a i o he da a wi h a wo-s a e unc ion (Supplemen a y Figu e S4). In composi e, hese expe imen s indica e ha wi hin he 0–5 pN o ce ange, he FECs e lec he in insic mechan- ical p ope ies o he p o ein–DNA complexes a each ex- pe imen al condi ion. The absence o hys e esis wi hin his o ce ange, e en a sal ype and concen a ions p omo ing he o ma ion o seconda y s uc u e on ssDNA (Supple- men a y Figu e S2), suppo s he conclusion ha Hm SSB emo es all o mos o he sal -induced seconda y s uc u e om he ssDNA and ha he p o ein–DNA complexes a e a equilib ium. Elimina ion o sal -induced hys e esis o in- di idual, long ssDNA molecules by E. coli SSB binding has also been epo ed ecen ly (32). Sal and p o ein concen a ion modula e he Hm SSB bind- ing mode o p e o med ssDNA molecules To de e mine he speci ic o ganiza ion o he p o ein–DNA complexes assembled on p e o med ssDNA molecules based on he abo e esul s, we i he e e sible po ion in he 0–5 pN ange o all he FECs aken unde each expe imen- al condi ion (N ∼200, see ’Ma e ials and Me hods’ sec ion) wi h he ollowing model (49). Because o da e he e is no expe imen al e idence o coope a i e binding by Hm SSB o ssDNA, he model conside s ha he p o ein could load andomly along he DNA empla e and bo h co e ed and non-co e ed ssDNA segmen s may coexis along he poly- me . Wi hin he 0–5 pN o ce ange, he DNA ends en- e ing and exi ing he p o ein e ame a e expec ed o oc- cupy opposi e sides o he Hm SSB e ame and main ain hei o ien a ion along he pulling coo dina e (see below). In his case, in o de o compu e he polyme ex ension, i is possible o desc ibe an e ec i e p o ein–DNA com- plex as i i we e a mix u e o wo independen polyme s, as shown in Figu e 3A. The e o e, a equilib ium, he ex en- sion pe nucleo ide o he p o ein–DNA complex a each o ce, Xcomplex(F),is gi en by he sum o he ex ensions o he ssDNA co e ed by Hm SSB p o eins,X p o (F),plus any po ion o p o ein- ee ssDNA, XssDNA(F), weigh ed by he ssDNA co e age (δ): Xcomplex(F)=δXp o (F)+(1 −δ)XssDNA(F),(1) whe e δis he ac ion o nucleo ides bound by Hm SSB and (1 −δ) is he ac ion o ee nucleo ides.On one hand, o de e mine he con ibu ion o he p o ein- ee ssDNA e- gion, (1 −δ)XssDNA(F), o he inal ex ension o he nucleo- p o ein complex, he a e age ex ension pe nucleo ide o he ee ssDNA, XssDNA(F), was ob ained di ec ly om he ex- pe imen al FECs o he ssDNA measu ed unde each expe - imen al condi ion (Supplemen a y Figu e S2). In his way he e ec o sal ype and concen a ion on XssDNA(F)was conside ed a he ele an o ce ange (Supplemen a y Fig- u e S2). On he o he hand, he con ibu ion o he p o ein- co e ed egion o he inal ex ension was modeled using he FJC o polyme elas ici y (46). The FJC model assumes ha each Hm SSB e ame –DNA complex beha es as a igid od, wi h a Kuhn leng h, Lends, equal o he end- o-end dis ance o he ssDNA w apped a ound a e ame . Then, he ex ension pe nucleo ide o he p o ein-co e ed egion is desc ibed as: Xp o (F)=Lends Nn co h FL ends kBT−kBT FL ends ,(2) whe e Nn is he a e age numbe o nucleo ides occluded by each e ame and co h(FL ends/kBT)−kBT/FL ends is he Lange in unc ion o FL ends/kBTand accoun s o he a e age alignmen o he e ame s wi h espec o he di- ec ion o he ex e nal o ce (F), Figu e 3A. As shown in 7242 Nucleic Acids Resea ch, 2017, Vol. 45, No. 12 Figu e 3. Hm SSB binding modes o p e o med ssDNA molecules. (A) Schema ic ep esen a ion o he e ec i e o ganiza ion o Hm SSB–DNA complex (see main ex ); indi idual Hm SSB e ame s a e shown as a g oup o ou sphe es. The ex ension o he p o ein–DNA complex unde o ce, Xcomplex (F), is gi en by he sum o ex ensions o he p o ein-bound, δ·Xp o (F) and unbound, (1-␦)·XssDNA(F), sec ions o he complex (Equa ion 1),whe e δis he ac ion o ssDNA co e ed by he p o ein. Nn , blue line, is he a e age numbe o w apped nucleo ides pe e ame . Lends is he e ec i e physical size o an indi idual e ame –DNA complex, 5≤Lends≤8 nm. Fo a ixed Lends, i s o he equilib ium FECs o he p o ein–DNA complex wi h Equa ion (1) ende ed he alues o Nn and δ o all expe imen al sal and p o ein concen a ions. (B) Dependence o Nn (n / e ame ) on Hm SSB concen a ion ([SSB] nM) o all sal condi ions s udied (Lends=6 nm). (C) Dependence o Nn on he [SSB]/[NaCl] a io o Lends=6 nm. (D) Dependence o ssDNA co e age (δ) on he [SSB]/[NaCl] a io o Lends=6 nm. Fo (B), (C) and (D) e o ba s ep esen s.e. and ed symbols ep esen he da a ob ained o complexes assembled in he p esence o 50 mM NaCl plus 4 mM MgCl2(which a e ele an o Figu e 4). Two s a is ically di e en Nn alues p e ail, indica ing ha Hm SSB can bind o p eassembled ssDNA in a low (Hm SSBL, ligh blue a ea) and a high (Hm SSBH, ligh ed a ea) si e size binding modes. Colo ed a eas ep esen he si e sizes (and s anda d de ia ions) o each binding mode a e a e aging he si e sizes alues ob ained o each Lends (Table 1). Supplemen a y Figu e S7 shows he Nn and δ alues ob ained wi h Lends=5, 7 and 8 nm. Nucleic Acids Resea ch, 2017, Vol. 45, No. 12 7243 he p e ious sec ion, mechanical ension a ec s e y sim- ila ly he o e all o ganiza ion o ssDNA a ound EcoSSB and Hm SSB e ame s. Based on his expe imen al obse - a ion, we conside ed ha wi hin he o ce ange s udied (0– 5 pN) he DNA ends occupy opposi e sides o he Hm SSB e ame , as epo ed in a ecen o ce spec oscopy s udy on a single EcoSSB-(dT)70 complex (33)(Figu e3A). The e- o e, he alue o Lends should be wi hin he c ys allog aphic size o he Hm SSB e ame , 5 ≤Lends ≤8nm(21). Fo a ixed Lends, he i s o he FECs ende ed he a e age numbe o nucleo ides w apped pe e ame (Nn ) and he p o ein co e age, δ, o all measu ed expe imen al condi ions. Fi s o indi idual FECs a e shown in Supplemen a y Figu e S5. In e es ingly, o each Lends, wo s a is ically di e en Nn alues p e ailed depending on sal and p o ein con- cen a ions, indica ing ha Hm SSB can bind o p eassem- bled ssDNA in a leas wo di e en binding modes; a low si e-size, Hm SSBLand a high si e-size, Hm SSBH, bind- ing modes (Figu e 3B and C, Supplemen a y Figu e S6 and Table S1). Simila o he beha io desc ibed o he EcoSSB (7,50,51), he low si e-size binding mode domina es a he lowes NaCl concen a ions s udied (10 mM) and/o highes [SSB]/[NaCl] a ios ([SSB]/[NaCl] >0.5–1·106), while he high si e-size mode p e ails a he highes NaCl concen a ions (300 mM) and/o lowes [SSB]/[NaCl] a- ios ([SSB]/[NaCl] <0.5–1·106). A in e media e and nea physiological sal concen a ions he binding mode depends mainly on he Hm SSB concen a ions, wi h he Hm SSBL mode p e ailing a p o ein concen a ions highe han ∼50– 100 nM. The e o e, as expec ed o a p o ein ha can bind o a long ssDNA in wo modes, he lowe si e-size mode is a o ed a high p o ein-binding densi ies, because mo e p o ein can be bound o he ssDNA in his mode. We no e ha he a e age numbe o nucleo ides w apped pe e ame (Nn ) in each binding mode depends on he Lends alues conside ed in he i s (Figu e 3B and Supplemen- a y Figu e S7). All possible alues o Nn in each bind- ing mode a e summa ized in Table 1. Acco ding o ou e- sul s, on a e age and o all Lends, Hm SSB binds ∼30% ewe nucleo ides pe e ame in he Hm SSBLbinding mode han in he Hm SSBHbinding mode. The a e age alues ob ained o Nn a e compa ible o he appa en binding-si e sizes on poly(dT) oligonucleo ides as a unc- ion o sal concen a ion desc ibed p e iously in bulk s ud- ies o Hm SSB (50 and 70 n / e ame ) (23), D osophila melanogas e m SSB (68–120 n / e ame ) (52) and EcoSSB (35 and 65 n / e ame ) (50)(Table1). Ou da a also show ha a he highes [SSB]/[NaCl] a- ios he Hm SSB occupies ≥90% o he ssDNA molecule and i is hus, expec ed o o m long p o ein clus e s along he DNA unde condi ions in which binding in he Hm SSBLmode p e ails (Figu e 3D). By con as , he p o- ein co e age dec eases o ∼70% a he lowes [SSB]/[NaCl] a ios in which he Hm SSBHbinding mode domina es (Figu e 3D). In con as o Nn , p o ein co e age does no signi ican ly change wi h he Lends alues conside ed in he i s (Supplemen a y Figu e S7). Table 1. A e age numbe o nucleo ides w apped pe e ame in each binding mode o di e en Lends alues Fo all expe imen al condi ions, he uppe and lowe ows show he a - e age si e sizes (n / e ame ) co esponding o all da a aken abo e and below [SSB]/[NaCl] =0.5–1·106, espec i ely (Figu e 3C and Supplemen- a y Figu e S7). E o shows s anda d de ia ions. Fo each Lends alue, when binding is assayed on p eassembled ssDNA he a e age si e-sizes a e s a is ically di e en , ep esen ing he low (Hm SSBL, ligh blue) and he high (Hm SSBH, ligh ed) si e-sizes binding modes. Ins ead, o each Lends alues, when binding occu s du ing DNA syn hesis he a e age si e-sizes a e no s a is ically di e en , and a e ully compa ible wi h he low si e-size binding mode. A de ailed s a is ical analysis o he di e ences be ween he epo ed si e sizes is shown in Supplemen a y Table S1. Co- eplica ional binding o Hm SSB o ssDNA p omo es he low si e-size binding mode o all expe imen al condi ions s udied Mo i a ed by he pi o al ole o Hm SSB du ing m DNA eplica ion, we designed a se o expe imen s o de e mine whe he a speci ic Hm SSB binding mode is used p e e en- ially when he ssDNA is g adually gene a ed in he 5–3 di ec ion du ing he p ocess o DNA syn hesis. Fo hese expe imen s we used he DNA hai pin con aining he ds- DNA handle, which p o ides a ee 3-end o DNA poly- me ase loading, modi ied wi h an abasic si e a he loop end (Figu e 4A and see ’Ma e ials and Me hods’ sec ion). Ex- cep o he abasic modi ica ion, his is he same hai pin we used o measu e Hm SSB binding o p eassembled ssDNA in he p esence o MgCl2(50 mM NaCl/4mMMgCl 2). Fo eplica ion o he hai pin we chose he bac e iophage Phi29 DNA polyme ase because i s p ope ies as a hy- b id polyme ase-helicase (53,54) allows syn hesis h ough he ull leng h o he hai pin wi hou dissocia ion, un il i eaches he abasic si e loca ed a he end loop, which in- hibi s u he eplica ion o he displaced s and (43)(Fig- u e 4A and Supplemen a y Figu e S8). S and displacemen syn hesis was pe o med a 50 mM NaCl/4mMMgCl 2 and inc easing Hm SSB concen a ions (5, 10, 50 and 200 nM), a o ces ∼3 pN. A e comple ion o DNA syn he- sis and be o e measu ing he esul ing FECs he excess o ee Hm SSB in solu ion was washed ou o he low cell. In e es ingly, indi idual DNA syn hesis ajec o ies did no show signi ican pauses and/o sudden dis ance changes, ei he wi h o wi hou Hm SSB in solu ion, sugges ing a g adual binding o Hm SSB o he ssDNA as i is eleased a an a e age a e o ∼30 n /s(Figu e4B and Supplemen- a y Figu e S8). The inal p oduc o he eac ion is a hyb id DNA molecule con aining 5226 bp o dsDNA and 2540 n o ssDNA ha becomes co e ed g adually by he Hm SSB. Rep esen a i e FECs o p o ein–DNA complexes ob ained 7244 Nucleic Acids Resea ch, 2017, Vol. 45, No. 12 Figu e 4. Co- eplica ional binding o Hm SSB o ssDNA. (A) Schema ic o he expe imen al se -up. The DNA hai pin con aining a dsDNA handle (2686 bp) was modi ied wi h an abasic si e a he loop apex ( ed s a ) and a ached be ween he beads in he op ical weeze s (see ’Ma e ials and Me hods’ sec ion). S and displacemen DNA syn hesis ac i i ies we e pe o med using he Phi29 DNA polyme ase (g een iangle) and ini ia ed a F∼3pN in he p esence o a ying Hm SSB (b own e ame ) concen a ions. As eplica ion p oceeds he displaced ssDNA s and is g adually co e ed by he Hm SSB. S and displacemen syn hesis e mina es a he abasic si e, yielding a hyb id DNA molecule wi h 5226 bp o dsDNA and 2540 n o ssDNA g adually co e ed by he Hm SSB (see also Supplemen a y Figu e S8). A e sub ac ion o he dsDNA con ibu ion (Supplemen a y Figu e S3), i s wi h Equa ion (1) o he equilib ium FECs o he p o ein–DNA complexes ob ained unde hese condi ions ende ed, o a ixed Lends, he alues o he a e age numbe o w apped nucleo ides pe e ame , Nn and he ac ion o ssDNA co e ed by he p o ein (δ) o all Hm SSB concen a ions. (B) Independen eplica ion aces wi hou (black) and wi h (g een) Hm SSB in solu ion (50 nM). Each ace ep esen s he eal- ime change in dis ance (∼30 n /s) associa ed wi h he ull eplica ion o he hai pin a F∼3pN.(C) Dependence o Nn on Hm SSB concen a ion o Lends=6 nm. Colo ed a eas ep esen he app oxima ed a e age si e sizes (see Figu e 3). (D) Dependence o ssDNA co e age (δ) on Hm SSB concen a ion o Lends=6 nm. Fo (C) and (D) g een symbols ep esen he Nn and δ alues ob ained o p o ein–DNA complexes assembled du ing DNA syn hesis and ed symbols ep esen he Nn and δ alues o complexes assembled on p e o med ssDNA. Fo bo h condi ions he sal concen a ion was 50 mM NaCl/4mMMgCl 2and he Hm SSB concen a ion 5, 10, 50 100 o 200 nM. Fo all plo s e o ba s ep esen s.e. See Supplemen a y Figu e S7 o Nn and δ alues ob ained wi h Lends =5, 7 and 8 nm. unde hese condi ions a e shown in Supplemen a y Figu e S8. Fo hese expe imen s, he ex ension pe nucleo ide o he ssDNA p o ein-co e ed egion a each o ce (Xcomplex (F)in Equa ion 1) was ob ained by sub ac ing om he o al ex- ension o he molecule he ex ension o he dsDNA po - ion a each o ce (Supplemen a y Figu e S3). As desc ibed abo e, o a ixed Lends, i s wi h Equa ion (1) o he 0–5 pN egion o hese FECs ende ed he a e age numbe o nu- cleo ides w apped pe e ame (Nn ) and he p o ein co - e age (δ) o all p o ein concen a ions. In sha p con as o he esul s ob ained unde he same p o ein concen a- ions and sal ype (4 mM MgCl2and 50 mM NaCl) on p eassembled ssDNA, he a e age numbe o nucleo ides w apped pe e ame (Nn ) and he p o ein co e age (δ) do no change wi h he p o ein concen a ion. Ins ead, in all cases, Hm SSB binds p e e en ially in he low si e-size binding mode, Hm SSBL, co e ing almos en i ely he e- leased ssDNA (δ∼95%, Figu e 4C and D). Iden ical be- ha io s we e obse ed o Lends alues be ween 5 and 8 nm (Supplemen a y Figu e S7). The a e age binding si e-sizes ob ained o each Lends a e summa ized in Table 1.These esul s indica e ha he unidi ec ional and g adual elease o ssDNA a a a e o ∼30 n /s ul ima ely de e mines he w apping mode o Hm SSB along he nascen ssDNA a he expe imen al condi ions unde s udy. Nucleic Acids Resea ch, 2017, Vol. 45, No. 12 7245 Figu e 5. Hm SSB–ssDNA binding ene gy. Gibbs ene gy equi ed o un- w ap 1 n om each Hm SSB e ame as a unc ion o NaCl concen a ion (mM), ΔGw(in kBT/n , see ’Ma e ials and Me hods’ sec ion), o 5 (black), 50 (blue), 100 (magen a) and 200 (cyan) nM Hm SSB. Red line ep esen s a linea i o he da a aken a 10, 50, 100 and 300 mM NaCl (R2=0.84, slope =−0.001 ±0.0001 kBT/(n ·M)). Red and g een symbols show he ΔGw alues o p o ein–DNA complexes assembled a 50 mM NaCl plus 4 mM MgCl2on p e o med ssDNA ( ed) o du ing DNA eplica ion (g een) a 5, 10, 50 and 200 nM Hm SSB concen a ions. E o ba s ep esen s.e. Hm SSB–ssDNA binding ene gy The FECs e lec he wo k equi ed o ex end he p o ein– DNA complexes, and hus can be used o calcula e he Hm SSB–ssDNA w apping ene gy. To calcula e he a e age Gibbs ene gy equi ed o unw ap 1 n om each e ame (ΔGw), he a ea be ween he p o ein-bound and he elaxing ee ssDNA FECs was di ided by he o al numbe o nu- cleo ides, and co ec ed by he p o ein co e age alues cal- cula ed p e iously o each expe imen al condi ion. The e- laxing cu e was used ins ead o he s e ching ee ssDNA cu e o a oid he ene ge ic con ibu ion o long- ange ss- DNA seconda y s uc u e induced by sal (32) (Supplemen- a y Figu e S2). Unde ou expe imen al condi ions (pH = 7.5 and 22◦C) and wi hin ou expe imen al e o , ΔGwdoes no depend on Hm SSB concen a ion (Supplemen a y Fig- u e S9). Howe e , ΔGwdec eases linea ly wi h NaCl con- cen a ions om ΔGw∼0.7 ±0.1 kBT/n a 10 mM NaCl o 0.4 ±0.1 kBT/n a 300 mM NaCl (Figu e 5). In e es ingly, he p esence o 4 mM MgCl2(50 mM NaCl plus 4 mM MgCl2) dec eases ΔGwby ∼30%, ΔGw∼0.45 kBT/n (Figu e 5). The dec ease o ΔGwwi h sal concen a- ion and ype highligh s he impo ance o elec os a ic in- e ac ions o s abilize he Hm SSB–ssDNA complexes and e lec s he highe a ini y o Mg2+ o e Na+ions o he ssDNA. The ΔGw alues ound o Hm SSB (ΔGw∼0.4– 0.7 kBT/n ) a e wi hin he ange bu signi ican ly highe han hose epo ed o EcoSSB p o ein unde simila ex- pe imen al condi ions, whe e ΔGwis ∼0.13–0.42 kBT/n (32–34). These di e ences may be due o an inc eased num- be o a oma ic esidues in he Hm SSB p o ein (16 o he Hm SSB e sus 13 o he EcoSSB), which may a o hy- d ophobic in e ac ions wi h ssDNA bases and/o he ac ha he mi ochond ial p o ein lacks he lexible acidic C- e minus o EcoSSB, which may p o ide addi ional s e ic hind ance and elec os a ic epulsion o ssDNA binding by EcoSSB (5). Howe e , as binding a ini ies can only be meaning ully compa ed unde iden ical solu ion condi ions (7), we canno ule ou ha he obse ed di e ences in ΔGw be ween he wo p o eins may a ise om he di e en expe - imen al condi ions used in each s udy. We did no obse e signi ican di e ences in ΔGw o ei he binding mode (Sup- plemen a y Figu e S9). DISCUSSION We used op ical weeze s and heo e ical modeling o in- e oga e he s uc u e and ene ge ics o di e en binding modes o Hm SSB o long, p e o med ssDNA molecules and o de e mine he p e ailing binding mode when p o- ein binding is coupled o a DNA me abolic p ocess, such as DNA eplica ion, in which g adual ssDNA gene a ion and p o ein binding occu concomi an ly. Despi e he unce ain y o he end- o-end dis ance o he DNA w apped a ound each e ame (5 nm ≤Lends ≤8 nm), ou da a indica es clea ly ha Hm SSB p o ein binds p e e en ially in a leas wo s a is ically di e en modes o long, p e o med ssDNA molecules depending on he p o- ein and sal concen a ion (Table 1and Supplemen a y Ta- ble S1): he low si e-size binding mode o Hm SSBLand he high si e-size binding mode o Hm SSBH.Ona e age, he Hm SSBLbinding mode binds ∼30% ewe nucleo ides pe e ame han he Hm SSBHbinding mode. We no e ha a condi ions close o whe e he ansi ion be ween modes oc- cu s, we canno disce n he coexis ence o he wo binding modes along he same ssDNA molecule (i.e. [SSB]/[NaCl]∼ 0.5–1·10−6, Figu e 3C and Supplemen a y Figu e S6). In e es ingly, he sal and p o ein concen a ion depen- dencies ound in ou wo k o he Hm SSBLbinding mode a e s ikingly simila o hose desc ibed o he EcoSSB low si e-size binding mode (EcoSSB35), sugges ing ha he Hm SSBLmay be he s uc u al equi alen o he EcoSSB35 mode. Fo example, Hm SSBL, p e ailed a he lowes NaCl concen a ions (10 mM) and a he highes p o ein bind- ing densi ies as desc ibed p e iously o EcoSSB35 (2,7). In con as , Hm SSBHbinding mode ound in ou wo k p e- ails a he highes NaCl concen a ions (300 mM) and a he lowes p o ein binding densi ies, indica ing ha i may be he s uc u al equi alen o he EcoSSB high si e-size binding mode (EcoSSB65 o EcoSSB56), which p e ails a iden ical sal and p o ein concen a ions (2,7). In ac , i he lowes Lends alue (Lends=5 nm) we e o e lec he ac ual s uc u al o ganiza ion o he p o ein–DNA complex (as explained in Supplemen a y Figu e S7), he a e age bind- ing si e sizes expec ed o Hm SSBLand Hm SSBH,∼31 and 53 n / e ame , espec i ely, would be ema kably sim- ila o hose desc ibed o EcoSSB35 and EcoSSB56 (Table 1). We no e ha as o ce is likely shi ing he DNA ends owa d he opposi e sizes o he e ame (33), he ac ual a e age numbe o nucleo ides w apped pe e ame in he absence o o ce would p obably be highe han epo ed o bo h binding modes. Ou da a also indica es ha a condi ions a o ing he lowe si e-size binding mode, Hm SSBL, he p o ein co - e s ∼90% o ssDNA, which is abou 20% mo e han un- de condi ions a o ing he highe si e-size binding mode