Mutagenic Bypass of an Oxidized Abasic Lesion-Induced DNA Interstrand Cross-Link Analogue by Human Translesion Synthesis DNA Polymerases

Abstract

5′-(2-Phosphoryl-1,4-dioxobutane) (DOB) is an oxidized abasic site that is produced by several antitumor agents and γ-radiolysis. DOB reacts reversibly with a dA opposite the 3′-adjacent nucleotide to form DNA interstrand cross-links (ICLs), genotoxic DNA lesions that can block DNA replication and transcription. Translesion synthesis (TLS) is an important step in several ICL repair pathways to bypass unhooked intermediates generated by endonucleolytic incision. The instability of DOB-ICLs has made it difficult to learn about their TLS-mediated repair capability and mutagenic potential. We recently developed a method for chemically synthesizing oligonucleotides containing a modified DOB-ICL analogue. Herein, we examined the capabilities of several highly relevant eukaryotic TLS DNA polymerases (pols), including human pol η, pol κ, pol ι, pol ν, REV1, and yeast pol ζ, to bypass this DOB-ICL analogue. The prelesion, translesion, and postlesion replication efficiency and fidelity were examined. Pol η showed moderate bypass activity when encountering the DOB-ICL, giving major products one or two nucleotides beyond the cross-linked template nucleotide. In contrast, DNA synthesis by the other pols was stalled at the position before the cross-linked nucleotide. Steady-state kinetic data and liquid chromatography–mass spectrometry sequencing of primer extension products by pol η unambiguously revealed that pol η-mediated bypass is highly error-prone. Together, our study provides the first set of in vitro evidence that the DOB-ICL is a replication-blocking and highly miscoding lesion. Compared to several other TLS pols examined, pol η is likely to contribute to the TLS-mediated repair of the DOB-ICL in vivo.

Bypass of DNA Interstrand crosslinks by a Rev1-DNA polymerase ζ complex

“…From an enzymatic perspective, Pol h would be an ideal enzyme to serve as an inserter for ICL. In previous studies, Pol h has been shown to replicate past several model ICL lesions (33,49,50), and this is confirmed in the current study. Interestingly, in both the previous and our current studies, Pol h has the propensity to stall either after insertion at ICL, or just past the lesion (Fig.…”

O6-2′-Deoxyguanosine-butylene-O6-2′-deoxyguanosine DNA Interstrand Cross-Links Are Replication-Blocking and Mutagenic DNA Lesions

“…32,46,47 By contrast, pol κ has minimal bypass activities toward major groove ICLs ( N 7-dG- N 7-dG) derived from cisplatin, nitrogen mustard, 48 and oxidized abasic lesion. 30 Consistent with these properties of pol κ , our results demonstrate that major groove adducts O 6 -dG-C4- O 6 -dG ICLs are poor substrates for the TLS activity of pol κ (Figures 3–5, Tables S1 and S2). Together, these results suggest that the catalytic activity of pol κ is unlikely to contribute to replication-dependent repair of O 6 -dG ICLs in vivo .…”

“…49,50 In addition, pol η is able to carry out insertion as well as extension with a number of structurally distinct ICLs, including those induced by acrolein, 51 cisplatin, 48 nitrogen mustards, 48,52 and oxidized abasic sites. 30 In the present study, pol η showed the highest bypass activity toward O 6 -dG-C4- O 6 -dG ICL substrates compared to the other three DNA pols. The clear difference observed in bypass activities using cell extracts from human XP-V fibroblasts and those complemented with pol η suggests that pol η is important for rescuing a stalled replication fork in the replication-dependent ICL repair pathway (Figure 7).…”

“…This powerful technique is complementary to the kinetic analysis and is particularly suitable for detecting insertions/deletions (indels). 30,38 …”

Involvement of translesion synthesis DNA polymerases in DNA interstrand crosslink repair

“…3) [21, 22, 67]. Interestingly, the pattern of bypass by Pol η is similar for various ICLs, stalling predominantly at 0, +1 and +2 positions and the structure-function relationships mentioned above apply in particular to studies with Pol η. Interestingly, no stalling was observed at all when the duplex around an ICL is reduced to a single crosslinked nucleotide, emphasizing the dramatic influence of the unhooking step on polymerase activity on ICLs [23].…”