A non‐proteolytic release mechanism for HMCES‐DNA‐protein crosslinks

Abstract

The conserved protein HMCES crosslinks to abasic (AP) sites in ssDNA to prevent strand scission and the formation of toxic dsDNA breaks during replication. Here, we report a non‐proteolytic release mechanism for HMCES‐DNA‐protein crosslinks (DPCs), which is regulated by DNA context. In ssDNA and at ssDNA‐dsDNA junctions, HMCES‐DPCs are stable, which efficiently protects AP sites against spontaneous incisions or cleavage by APE1 endonuclease. In contrast, HMCES‐DPCs are released in dsDNA, allowing APE1 to initiate downstream repair. Mechanistically, we show that release is governed by two components. First, a conserved glutamate residue, within HMCES' active site, catalyses reversal of the crosslink. Second, affinity to the underlying DNA structure determines whether HMCES re‐crosslinks or dissociates. Our study reveals that the protective role of HMCES‐DPCs involves their controlled release upon bypass by replication forks, which restricts DPC formation to a necessary minimum.

Formation and Properties of DNA Adducts Generated by Reactions of Abasic Sites with 1,2-Aminothiols Including Cysteamine, Cysteine Methyl Ester, and Peptides Containing N-Terminal Cysteine Residues

“…These proteins rapidly capture AP sites located at single-strand/duplex junctions in DNA via rapid thiazolidine formation. These AP–protein adducts are resistant to strand cleavage reactions that otherwise might induce genomic instability. ,,,, After repair synthesis restores the AP–protein adduct to the context of double-stranded DNA, the protein catalyzes its own dissociation from the DNA, leaving the AP site to be repaired via the base excision repair pathway. ,, The slow strand cleavage reactions that we observe for the thiazolidine adducts, with half-lives in the range of 12–60 h, are unlikely to be relevant to the properties of the SRAP proteins because these DNA–protein adducts are relatively short-lived, , and the microenvironment surrounding the thiazolidine residue in the active site of these proteins disfavors strand cleavage …”

Novel Processes Associated with the Repair of Interstrand Cross-Links Derived from Abasic Sites in Duplex DNA: Roles for the Base Excision Repair Glycosylase NEIL3 and the SRAP Protein HMCES

“…74−77,79,83−85 Paulin et al used AP-oligonucleotide exchange reactions to show that DPC formation in single-stranded by HMCES and its bacterial orthologue YedK is a reversible process. 76,78,82 In accordance with Tolman's principle of microscopic reversibility (1925), dissociation of the YedK-AP DPC, like the formation process, is self-catalyzed by the active site residue Glu105 and, to a lesser extent, His160. 76 It was subsequently shown that reversal of the HMCES-AP DPC is similarly dependent upon catalysis by Glu127 and, to a lesser degree, His210, as well as the DNA-binding properties of the protein.…”

“…78 Formation of the DPC by E127A and H210A mutants of HMCES occurs somewhat more slowly than the wild-type protein but is nonetheless complete within 10 min. 78 Formation of the YedK-AP DPC in single-stranded DNA, similarly, is a fast reaction. 76 The HMCES-AP DPC in single-stranded DNA is stable for hours and protects AP sites against endonucleolytic cleavage by APE1.…”

“…76 It was subsequently shown that reversal of the HMCES-AP DPC is similarly dependent upon catalysis by Glu127 and, to a lesser degree, His210, as well as the DNA-binding properties of the protein. 78,82 Importantly, hybridization of a complementary strand to the HMCES-AP DPC in single-stranded DNA dramatically destabilizes the cross-link attachment, leading to nearly complete dissociation within 1 h. 78,79,82 Again, rapid dissociation of the HMCES-AP DPC in duplex DNA was completely reliant on self-catalysis involving Glu127. 78 Studies in human cell lines provided evidence that selfcatalyzed reversal of the HMCES-AP cross-link is an important mechanism for resolution of the DPC.…”

“…78,82 Importantly, hybridization of a complementary strand to the HMCES-AP DPC in single-stranded DNA dramatically destabilizes the cross-link attachment, leading to nearly complete dissociation within 1 h. 78,79,82 Again, rapid dissociation of the HMCES-AP DPC in duplex DNA was completely reliant on self-catalysis involving Glu127. 78 Studies in human cell lines provided evidence that selfcatalyzed reversal of the HMCES-AP cross-link is an important mechanism for resolution of the DPC. 82 These experiments showed that resolution of the HMCES-AP adducts generated at replication forks was not strongly dependent upon proteolytic degradation by either SPRTN or the proteasome but was significantly delayed in cells expressing the catalytically compromised E127Q mutant of HMCES.…”