Accurate and Efficient Bypass of 8,5′-Cyclopurine-2′-Deoxynucleosides by Human and Yeast DNA Polymerase η

Abstract

Reactive oxygen species (ROS), which can be produced during normal aerobic metabolism, can induce the formation of tandem DNA lesions, including 8,5'-cyclo-2'-deoxyadenosine (cyclo-dA) and 8,5'-cyclo-2'-deoxyguanosine (cyclo-dG). Previous studies have shown that cyclo-dA and cyclo-dG accumulate in cells and can block mammalian RNA polymerase II and replicative DNA polymerases. Here, we used primer extension and steady-state kinetic assays to examine the efficiency and fidelity for polymerase η to insert nucleotides opposite, and extend primer past, these cyclopurine lesions. We found that Saccharomyces cerevisiae and human polymerase η inserted 2'-deoxynucleotides opposite cyclo-dA, cyclo-dG, and their adjacent 5' nucleosides at fidelities and efficiencies that were similar as their respective undamaged nucleosides. Moreover, the yeast enzyme exhibited similar processivity in DNA synthesis on templates housing a cyclo-dA or cyclo-dG as those carrying an unmodified dA or dG; the human polymerase, however, dissociated from the primer-template complex after inserting one or two additional nucleotides after the lesion. Pol η's accurate and efficient bypass of cyclo-dA and cyclo-dG indicate that this polymerase is likely responsible for error-free bypass of these lesions, whereas mutagenic bypass of these lesions may involve other translesion synthesis DNA polymerases. Together, our results suggested that pol η may have an additional function in cells, i.e., to alleviate the cellular burden of endogenously induced DNA lesions, including cyclo-dA and cyclo-dG.

Mutagenicity and Genotoxicity of (5′S)-8,5′-Cyclo-2′-deoxyadenosine in Escherichia coli and Replication of (5′S)-8,5′-Cyclopurine-2′-deoxynucleosides in Vitro by DNA Polymerase IV, Exo-Free Klenow Fragment, and Dpo4

“…13 In contrast, our results with KF (exo – ), pol IV, and Dpo4 indicate that the cyclopurine lesions are very strong blocks of replication. This difference can be rationalized when a unique characteristic of pol η, part of which may also be shared by the E. coli pol V, 33 is considered.…”

Translesion Synthesis of 8,5′-Cyclopurine-2′-deoxynucleosides by DNA Polymerases η, ι, and ζ

“…In addition, depletion of Pol in human cells conferred a considerable reduction in mutation frequency of S-cdA, suggesting that Pol may be involved in error-prone replicative bypass of this lesion in mammalian system. This result is somewhat surprising, as our previous biochemical studies have sug- gested a role for Pol in the error-free nucleotide insertion opposite cPu lesions (31). The difference in the fidelity of lesion bypass by Pol between in vitro and in vivo studies may be attributed to two factors.…”

“…B and C, relative efficiencies (with respect to the corresponding damage-free substrates) of nucleotide incorporation across and past S-cdA (B) and S-cdG (C) by human Pol , , and , and a two-subunit yeast Pol complex (REV3/REV7). The efficiencies of Pol -mediated nucleotide insertion opposite the lesion site or the neighboring 5Ј nucleotide of S-cdA or S-cdG were published previously (31).…”

“…The aqueous portion was dried with Speed-Vac, desalted with HPLC, and dissolved in 12 l of water. The ODN mixture was subjected to LC-MS/MS analysis, as described elsewhere (18,25,31,38). An LTQ linear ion trap mass spectrometer (Thermo Electron) was set up for monitoring fragmentation of the [M-3H] 3Ϫ ions of the 13-mer ODNs (d(CATGGCGMGCTGT), where "M" designates A, T, C, or G).…”

“…However, it is still unclear how these cPu lesions compromise DNA replication in mammalian cells. In addition, although previous biochemical studies have suggested a role for Pol in the TLS across the cPu lesions in vitro (30,31), it remains unknown whether Pol is involved in the replicative bypass of cPu lesions in mammalian cells, and much less is known about whether other TLS polymerases, including Pol , Pol , and Pol , participate in the TLS across cPu lesions in vitro and in mammalian cells.…”