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.

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In yeast, dNTP pools expand drastically during DNA damage response. We show that similar dNTP elevation occurs in strains, in which intrinsic replisome defects promote the participation of error-prone DNA polymerase ζ (Polζ) in replication of undamaged DNA. To understand the significance of dNTP pools increase for Polζ function, we studied the activity and fidelity of four-subunit Polζ (Polζ4) and Polζ4-Rev1 (Polζ5) complexes in vitro at ‘normal S-phase’ and ‘damage-response’ dNTP concentrations. The presence of Rev1 inhibited the activity of Polζ and greatly increased the rate of all three ‘X-dCTP’ mispairs, which Polζ4 alone made extremely inefficiently. Both Polζ4 and Polζ5 were most promiscuous at G nucleotides and frequently generated multiple closely spaced sequence changes. Surprisingly, the shift from ‘S-phase’ to ‘damage-response’ dNTP levels only minimally affected the activity, fidelity and error specificity of Polζ complexes. Moreover, Polζ-dependent mutagenesis triggered by replisome defects or UV irradiation in vivo was not decreased when dNTP synthesis was suppressed by hydroxyurea, indicating that Polζ function does not require high dNTP levels. The results support a model wherein dNTP elevation is needed to facilitate non-mutagenic tolerance pathways, while Polζ synthesis represents a unique mechanism of rescuing stalled replication when dNTP supply is low.

Abstract DNA polymerase ζ (Pol ζ) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases η, ζ and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based interstrand crosslink (ICL) with an 8-atom linker between the crosslinked bases. The Rev1–Pol ζ complex was most efficient in complete bypass synthesis, by 2–3 fold, compared to Pol ζ alone or Pol η. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol η, Pol ζ, and Rev1–Pol ζ. Rev1–Pol ζ, and particularly Pol ζ alone showed a tendency to stall before the ICL, whereas Pol η stalled just after insertion across the ICL. The stalling of Pol η directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol η providing a barrier to further elongation of the correct primer. No stalling by Rev1–Pol ζ directly past the ICL was observed, suggesting that the proposed function of Pol ζ as an extender DNA polymerase is also required for ICL repair.

DNA polymerase z (Pol z) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases h, z, and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based ICL with an 8-atom linker between the crosslinked bases. The Rev1-Pol z complex was most efficient in complete bypass synthesis, by 2-3 fold, compared to Pol z alone or Pol h. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol h, Pol z, and Rev1-Pol z. Rev1-Pol z, and particularly Pol z alone showed a tendency to stall before the ICL, whereas Pol h stalled just after insertion across the ICL. The stalling of Pol h directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol h providing a barrier to further elongation of the correct primer. No stalling by Rev1-Pol z directly past the ICL was observed, suggesting that the proposed function of Pol z as an extender DNA polymerase is also required for ICL repair.