Masatomi Shimizu

Erroneous incorporation of oxidized DNA precursors by Y-family DNA polymerases.

Deranged oxidative metabolism is a property of many tumour cells. Oxidation of the deoxynucleotide triphosphate (dNTP) pool, as well as DNA, is a major cause of genome instability. Here, we report that two Y‐family DNA polymerases of the archaeon Sulfolobus solfataricus strains P1 and P2 incorporate oxidized dNTPs into nascent DNA in an erroneous manner: the polymerases exclusively incorporate 8‐OH‐dGTP opposite adenine in the template, and incorporate 2‐OH‐dATP opposite guanine more efficiently than opposite thymine. The rate of extension of the nascent DNA chain following on from these incorporated analogues is only slightly reduced. These DNA polymerases have been shown to bypass a variety of DNA lesions. Thus, our results suggest that the Y‐family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis, in addition to facilitating translesion DNA synthesis. We also report that human DNA polymerase η, a human Y‐family DNA polymerase, incorporates the oxidized dNTPs in a similar erroneous manner.

Involvement of Y-Family DNA Polymerases in Mutagenesis Caused by Oxidized Nucleotides in Escherichia coli

Escherichia coli DNA polymerase IV incorporated 2-hydroxy-dATP opposite template guanine or thymine and 8-hydroxy-dGTP exclusively opposite adenine in vitro. Mutator phenotypes in sod/fur strains were substantially diminished by deletion of dinB and/or umuDC. DNA polymerases IV and V may be involved in mutagenesis caused by incorporation of the oxidized deoxynucleoside triphosphates.

Escherichia coli DNA polymerase III is responsible for the high level of spontaneous mutations in mutT strains.

Reactive oxygen species induce oxidative damage in DNA precursors, i.e. dNTPs, leading to point mutations upon incorporation. Escherichia coli mutT strains, deficient in the activity hydrolysing 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine 5′‐triphosphate (8‐oxo‐dGTP), display more than a 100‐fold higher spontaneous mutation frequency over the wild‐type strain. 8‐oxo‐dGTP induces A to C transversions when misincorporated opposite template A. Here, we report that DNA pol III incorporates 8‐oxo‐dGTP ≈ 20 times more efficiently opposite template A compared with template C. Single, double or triple deletions of pol I, pol II, pol IV or pol V had modest effects on the mutT mutator phenotype. Only the deletion of all four polymerases led to a 70% reduction of the mutator phenotype. While pol III may account for nearly all 8‐oxo‐dGTP incorporation opposite template A, it only extends ≈ 30% of them, the remaining 70% being extended by the combined action of pol I, pol II, pol IV or pol V. The unique property of pol III, a C‐family DNA polymerase present only in eubacteria, to preferentially incorporate 8‐oxo‐dGTP opposite template A during replication might explain the high spontaneous mutation frequency in E. coli mutT compared with the mammalian counterparts lacking the 8‐oxo‐dGTP hydrolysing activities.

Origins of Age-Related DNA Damage and Dietary Strategies for Its Reduction

The polyunsaturated fatty acids in biological membranes serve as both the target and source of oxidative damage and can be regarded as the most unstable class of biomolecules in the body. Lipid peroxides arising from both spontaneous and enzymatic oxidation of polyunsaturated fatty acids are the major source of endogenous DNA damage linked to various age-related pathologies and initiating carcinogenesis. Here we describe the major types of lipid peroxide-derived DNA adducts and propose a simple dietary strategy to reduce their formation. This may be particularly beneficial to the aging organism, which has progressively impaired natural protective systems.

Visualization of the interaction between archaeal DNA polymerase and uracil‐containing DNA by atomic force microscopy

Deamination of cytosine to uracil is a hydrolytic reaction that is greatly accelerated at high temperatures. The resulting uracil pairs with adenine during DNA replication, thereby inducing G:C to A:T transitions in the progeny. Interestingly, B‐family DNA polymerases from hyperthermophilic Archaea recognize the presence of uracil in DNA and stall DNA synthesis. To better understand the recognition mechanism, the binding modes of DNA polymerase B1 of Sulfolobus solfataricus (Pol B1) to uracil‐containing DNA were examined by gel mobility shift assays and atomic force microscopy. Although PolB1 per se specifically binds to uracil‐containing single‐stranded DNA, the binding efficiency was substantially enhanced by the initiation of DNA synthesis. Analysis by the atomic force microscopy showed a number of double‐stranded DNA (dsDNA) in the products of DNA synthesis. The generation of ds DNA was significantly inhibited, however, by the presence of template uracil, and intermediates where monomeric forms of Pol B1 appeared to bind to uracil‐containing DNA were observed. These results suggest that Pol B1 more efficiently recognizes uracil in DNA during DNA synthesis rather than during random diffusion in solution, and that single molecules of Pol B1 bind to template uracil and stall DNA synthesis.