A Rfa1-MN-based system reveals new factors involved in the rescue of broken replication forks

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 . 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