biostudies-literaturePalek MMinistry of HealthMEYSRVOGrant Agency of the Charles UniversityEuropean Union Next Generation EU7687-7703https://www.ebi.ac.uk/biostudies/studies/S-EPMC11260465biostudies-literatureUnknown52(13)RAD18 is an E3 ubiquitin ligase that prevents replication fork collapse by promoting DNA translesion synthesis and template switching. Besides this classical role, RAD18 has been implicated in homologous recombination; however, this function is incompletely understood. Here, we show that RAD18 is recruited to DNA lesions by monoubiquitination of histone H2A at K15 and counteracts accumulation of 53BP1. Super-resolution microscopy revealed that RAD18 localizes to the proximity of DNA double strand breaks and limits the distribution of 53BP1 to the peripheral chromatin nanodomains. Whereas auto-ubiquitination of RAD18 mediated by RAD6 inhibits its recruitment to DNA breaks, interaction with SLF1 promotes RAD18 accumulation at DNA breaks in the post-replicative chromatin by recognition of histone H4K20me0. Surprisingly, suppression of 53BP1 function by RAD18 is not involved in homologous recombination and rather leads to reduction of non-homologous end joining. Instead, we provide evidence that RAD18 promotes HR repair by recruiting the SMC5/6 complex to DNA breaks. Finally, we identified several new loss-of-function mutations in RAD18 in cancer patients suggesting that RAD18 could be involved in cancer development.Nucleic acids researchRAD18 directs DNA double-strand break repair by homologous recombination to post-replicative chromatin.PMC1126046535282268378050-KAV-NPUICZ.02.1.01/0.0/0.0/ 18_046/0016045NU20-03-00285LX22NPO5102LM2018129Kleibl ZChvojka SZemankova PVrtel RCerna MHejnalova MPalkova NKosarova MKratochvilova RKoudova MVocka MJedlickova NScheinost OJanikova MDuskova PForetova LZavoral TKrutilkova VMacurek LPalek MNovotny JMichalovska RCerna LTavandzis SMachackova EVacinova GHruskova LKleiblova PStranecky VSchwetzova DSubrt Iconsortium CZECANCAHorackova KNehasil PJanatova MVesela KVlckova ZSoukupova JHovhannisyan MHavranek OJelinkova SStastna BCurtisova VfalseRAD18 directs DNA double-strand break repair by homologous recombination to post-replicative chromatin.RAD18 is an E3 ubiquitin ligase that prevents replication fork collapse by promoting DNA translesion synthesis and template switching. Besides this classical role, RAD18 has been implicated in homologous recombination; however, this function is incompletely understood. Here, we show that RAD18 is recruited to DNA lesions by monoubiquitination of histone H2A at K15 and counteracts accumulation of 53BP1. Super-resolution microscopy revealed that RAD18 localizes to the proximity of DNA double strand breaks and limits the distribution of 53BP1 to the peripheral chromatin nanodomains. Whereas auto-ubiquitination of RAD18 mediated by RAD6 inhibits its recruitment to DNA breaks, interaction with SLF1 promotes RAD18 accumulation at DNA breaks in the post-replicative chromatin by recognition of histone H4K20me0. Surprisingly, suppression of 53BP1 function by RAD18 is not involved in homologous recombination and rather leads to reduction of non-homologous end joining. Instead, we provide evidence that RAD18 promotes HR repair by recruiting the SMC5/6 complex to DNA breaks. Finally, we identified several new loss-of-function mutations in RAD18 in cancer patients suggesting that RAD18 could be involved in cancer development.2024-01-01T00:00:00Z2024 Jul2025-04-04T14:46:28.116Z2025-04-04T14:46:28.116ZS-EPMC112604653888420210.1093/nar/gkae499