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

摘要

5'-(2-Phosphoryl-1,4-dioxobutane) (DOB) is an oxidized abasic site that is produced by several antitumor agents and gamma-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 endonudeolytic 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 oligonudeotides containing a modified DOB-ICL analogue. Herein, we examined the capabilities of several highly relevant eukaryotic TLS DNA polymerases (pols), including human pol eta, pol kappa, pol iota, pol nu, REV1, and yeast pol zeta to bypass this DOB-ICL analogue. The prelesion, translesion, and postlesion replication efficiency and fidelity were examined. Pol eta 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 eta unambiguously revealed that pol eta-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 eta is likely to contribute to the TLS-mediated repair of the DOB-ICL in vivo.