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The active site residues Gln55 and Arg73 play a key role in DNA damage bypass by S. cerevisiae Pol ?.

ABSTRACT: Eukaryotic DNA polymerase eta (Pol ?) plays a key role in the efficient and accurate DNA translesion synthesis (TLS) opposite UV-induced thymine dimers. Pol ? is also involved in bypass of many other DNA lesions but possesses low fidelity on undamaged DNA templates. To better understand the mechanism of DNA synthesis by Pol ? we investigated substitutions of evolutionary conserved active site residues Gln55 and Arg73 in Saccharomyces cerevisiae Pol ?. We analyzed the efficiency and fidelity of DNA synthesis by the mutant Pol ? variants opposite thymine dimers, abasic site, thymine glycol, 8-oxoguanine and on undamaged DNA. Substitutions Q55A and R73A decreased the catalytic activity and significantly affected DNA damage bypass by Pol ?. In particular, the Q55A substitution reduced the efficiency of thymine dimers bypass, R73A had a stronger effect on the TLS-activity opposite abasic site, while both substitutions impaired replication opposite thymine glycol. Importantly, the R73A substitution also increased the fidelity of Pol ?. Altogether, these results reveal a key role of residues Gln55 and Arg73 in DNA synthesis opposite various types of DNA lesions and highlight the evolutionary importance of the Pol ? TLS function at the cost of DNA replication accuracy.

SUBMITTER: Boldinova EO

PROVIDER: S-EPMC6037775 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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The active site residues Gln55 and Arg73 play a key role in DNA damage bypass by S. cerevisiae Pol η.

Boldinova Elizaveta O EO Ignatov Artem A Kulbachinskiy Andrey A Makarova Alena V AV

Scientific reports 20180709 1

Eukaryotic DNA polymerase eta (Pol η) plays a key role in the efficient and accurate DNA translesion synthesis (TLS) opposite UV-induced thymine dimers. Pol η is also involved in bypass of many other DNA lesions but possesses low fidelity on undamaged DNA templates. To better understand the mechanism of DNA synthesis by Pol η we investigated substitutions of evolutionary conserved active site residues Gln55 and Arg73 in Saccharomyces cerevisiae Pol η. We analyzed the efficiency and fidelity of D ...[more]

PMID: 29985422

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Project description:The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 microM, kcat 3960 min-1; Y51F mutant, Km 432 microM, kcat 6140 min-1; wild-type, Km 288 microM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (DeltaG) resulting from a smaller DeltaG of substrate binding. The side chain of Phe-42 acts as a phenyl 'cap' over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2. 08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 microM, kcat 14620 min-1; compared with values of 4.7 microM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a 'fast' to a 'slow' rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat 'fast', 760 (1620) min-1; kcat 'slow', 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole >10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid >30-fold more tightly than wild-type.

Dataset Information

The active site residues Gln55 and Arg73 play a key role in DNA damage bypass by S. cerevisiae Pol ?.

Publications

The active site residues Gln55 and Arg73 play a key role in DNA damage bypass by S. cerevisiae Pol η.

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