Journal Article

Mycobacterium smegmatis DinB2 misincorporates deoxyribonucleotides and ribonucleotides during templated synthesis and lesion bypass

Heather Ordonez and Stewart Shuman

in Nucleic Acids Research

Volume 42, issue 20, pages 12722-12734
Published in print November 2014 | ISSN: 0305-1048
Published online October 2014 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/gku1027

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Mycobacterium smegmatis DinB2 is the founder of a clade of Y-family DNA polymerase that is naturally adept at utilizing rNTPs or dNTPs as substrates. Here we investigate the fidelity and lesion bypass capacity of DinB2. We report that DinB2 is an unfaithful DNA and RNA polymerase with a distinctive signature for misincorporation of dNMPs, rNMPs and oxoguanine nucleotides during templated synthesis in vitro. DinB2 has a broader mutagenic spectrum with manganese than magnesium, though low ratios of manganese to magnesium suffice to switch DinB2 to its more mutagenic mode. DinB2 discrimination against incorrect dNTPs in magnesium is primarily at the level of substrate binding affinity, rather than kpol. DinB2 can incorporate any dNMP or rNMP opposite oxo-dG in the template strand with manganese as cofactor, with a kinetic preference for synthesis of an A:oxo-dG Hoogsteen pair. With magnesium, DinB2 is adept at synthesizing A:oxo-dG or C:oxo-dG pairs. DinB2 effectively incorporates deoxyribonucleotides, but not ribonucleotides, opposite an abasic site, with kinetic preference for dATP as the substrate. We speculate that DinB2 might contribute to mycobacterial mutagenesis, oxidative stress and quiescence, and discuss the genetic challenges to linking the polymerase biochemistry to an in vivo phenotype.

Journal Article.  8330 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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