The HMCES protein forms a covalent DNA-protein cross-link (DPC) with abasic (AP) sites in ssDNA, and the resulting HMCES-DPC is thought to suppress double-strand break formation in S phase. However, the dynamics of HMCES cross-linking and whether any DNA repair pathways normally include an HMCES-DPC intermediate remain unknown. Here, we show that an HMCES-DPC forms efficiently on the AP site generated during replication-coupled DNA interstrand cross-link (ICL) repair. We use this system to show that HMCES cross-links form on DNA after the replicative CMG helicase has passed over the AP site, and that HMCES is subsequently removed by the SPRTN protease. The HMCES-DPC suppresses DSB formation, slows translesion synthesis (TLS) past the AP site, and introduces a bias for insertion of deoxyguanosine opposite the AP site. These data show that HMCES-DPCs can form as constitutive intermediates in replication-coupled repair, and they suggest a general model of how HMCES protects AP sites during DNA replication. MainDNA abasic (AP) sites are common genomic lesions that arise from spontaneous DNA depurination/depyrimidination or as intermediates in the excision of damaged nucleobases by DNA glycosylases. When formed in double-stranded DNA, AP sites are usually incised by base excision repair factors (AP endonucleases and AP lyases), generating a one nucleotide gap that is filled in by DNA polymerase b 1 . AP sites are also susceptible to spontaneous cleavage through a b-elimination mechanism 2 . Importantly, enzymatic or spontaneous cleavage of AP sites located in ssDNA, e.g. during DNA replication, results in the formation of a DNA doublestranded break (DSB), which introduces the potential for gross chromosomal rearrangements. Recently, Cortez and colleagues demonstrated that the 5-hydroxymethylcytosine binding, embryonic stem cell-specific (HMCES) protein associates with replication forks, covalently cross-links to ssDNA AP sites, and suppresses accumulation of DSBs in cells treated with genotoxic agents that induce AP site formation 3 . Structural evidence indicates that the universally conserved SRAP domain of HMCES specifically cross-links to AP sites positioned in ssDNA in vitro through formation of a thiazolidine linkage 4-7 . Taken together, these observations indicate that HMCES stabilizes ssDNA AP sites during replication through formation of a DNAprotein cross-link (HMCES-DPC). HMCES-DPC formation also appears to suppress mutagenic bypass of AP sites by translesion synthesis (TLS). Loss of HMCES increases association of the REV1-Polz TLS polymerase with replication forks and increases the mutagenicity of UV-induced DNA damage 3,8 . Additionally, HMCES depletion slows replication forks upon induction of AP sites, and this defect is reversed by inhibition of REV1-Polz or depletion of the TLS polymerase Polk 9 .These data suggest that HMCES antagonizes AP site bypass by TLS polymerases and promotes a more rapid, error-free bypass mechanism, possibly involving template switching.

The HMCES protein forms a covalent DNA-protein cross-link (DPC) with abasic (AP) sites in ssDNA, and the resulting HMCES-DPC is thought to suppress double-strand break formation in S phase. However, the dynamics of HMCES cross-linking and whether any DNA repair pathways normally include an HMCES-DPC intermediate remain unknown. Here, we show that an HMCES-DPC forms efficiently on the AP site generated during replication-coupled DNA interstrand cross-link (ICL) repair. We use this system to show that HMCES cross-links form on DNA after the replicative CMG helicase has passed over the AP site, and that HMCES is subsequently removed by the SPRTN protease. The HMCES-DPC suppresses DSB formation, slows translesion synthesis (TLS) past the AP site, and introduces a bias for insertion of deoxyguanosine opposite the AP site. These data show that HMCES-DPCs can form as constitutive intermediates in replication-coupled repair, and they suggest a general model of how HMCES protects AP sites during DNA replication.