Eukaryotic Y-family polymerases bypass a 3-methyl-2 '-deoxyadenosine analog in vitro and methyl methanesulfonate-induced DNA damage in vivo

Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
Nucleic Acids Research (Impact Factor: 9.11). 05/2008; 36(7):2152-62. DOI: 10.1093/nar/gkn058
Source: PubMed


N3-methyl-adenine (3MeA) is the major cytotoxic lesion formed in DNA by SN2 methylating agents. The lesion presumably blocks progression of cellular replicases because the N3-methyl group hinders interactions between the polymerase and the minor groove of DNA. However, this hypothesis has yet to
be rigorously proven, as 3MeA is intrinsically unstable and is converted to an abasic site, which itself is a blocking lesion.
To circumvent these problems, we have chemically synthesized a 3-deaza analog of 3MeA (3dMeA) as a stable phosphoramidite
and have incorporated the analog into synthetic oligonucleotides that have been used in vitro as templates for DNA replication. As expected, the 3dMeA lesion blocked both human DNA polymerases α and δ. In contrast,
human polymerases η, ι and κ, as well as Saccharomyces cerevisiae polη were able to bypass the lesion, albeit with varying efficiencies and accuracy. To confirm the physiological relevance
of our findings, we show that in S. cerevisiae lacking Mag1-dependent 3MeA repair, polη (Rad30) contributes to the survival of cells exposed to methyl methanesulfonate
(MMS) and in the absence of Mag1, Rad30 and Rev3, human polymerases η, ι and κ are capable of restoring MMS-resistance to the normally MMS-sensitive strain.

Download full-text


Available from: PubMed Central · License: CC BY-NC
  • Source
    • "Specialized DNA polymerases such as polymerases η, ζ, ι, Rev1 and κ were identified to alleviate cell cycle termination caused by blockade of DNA replication [3] [4] [6] and by promoting error-free [7] [8] [9] [10] or error-prone [11] [12] [13] [14] [15] [16] [17] [18] DNA damage bypass due to a wide active site and an extra DNA binding domain [19]. DNA polymerase η (Pol η), while protecting cells against UV radiation [10], was found to replicate over DNA single-strand breaks (ssb), DNA mono-adducts like O 6 -methylguanine (O6-MeG), 3-methyl-adenine [15] [20] [21] [22] and intrastrand crosslinks induced by cisplatin [23] [24] [25]. TLS polymerase ζ (Pol ζ) also bypasses cisplatin adducts and adducts induced by the bulky therapeutic cisplatin analogs [24] [26] contributing to most mutations induced by DNAdamaging agents [4]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The importance of DNA polymerases in biology and biotechnology, and their recognition as potential therapeutic targets drives development of methods for deriving kinetic characteristics of polymerases and their propensity to perform polynucleotide synthesis over modified DNA templates. Among various polymerases, translesion synthesis (TLS) polymerases enable cells to avoid the cytotoxic stalling of replicative DNA polymerases at chemotherapy-induced DNA lesions, thereby leading to drug resistance. Identification of TLS inhibitors to overcome drug-resistance necessitates the development of appropriate high-throughput assays. Since polymerase-mediated DNA synthesis involves the release of inorganic pyrophosphate (PPi), we established a universal and fast method for monitoring the progress of DNA polymerases based on the quantification of PPi with a fluorescence-based assay that we coupled to in vitro primer extension reactions. The established assay has nanomolar detection limit in PPi and enables the evaluation of single nucleotide incorporation and DNA synthesis progression kinetics. The results demonstrated that the developed assay is a reliable method for monitoring TLS and identifying nucleoside and nucleotide-based TLS inhibitors. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Mar 2015 · Analytical Biochemistry
  • Source
    • "We first assessed the ability of DinB, in a binary complex with RecA, to replicate DNA using either an undamaged or alkylation lesion-containing template. The chemical structures of 2 0 -deoxyadenosine, the normal nucleoside; 2 0 - deoxy-3 0 -methyladenine, the fork-stalling alkylation lesion; and 2 0 -deoxyadenosine-3-deaza-3 0 -methyladenine, the stable analog generated by Plosky et al. (2008) are shown in Figure 1A. A schematic of the undamaged and 3-deaza-3- methyladenine-containing primer-templates are shown in Figure 1B for reference. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Alkylation DNA lesions are ubiquitous, and result from normal cellular metabolism as well as from treatment with methylating agents and chemotherapeutics. DNA damage tolerance by translesion synthesis DNA polymerases has an important role in cellular resistance to alkylating agents. However, it is not yet known whether Escherichia coli (E. coli) DNA Pol IV (DinB) alkylation lesion bypass efficiency and fidelity in vitro are similar to those inferred by genetic analyses. We hypothesized that DinB-mediated bypass of 3-deaza-3-methyladenine, a stable analog of 3-methyladenine, the primary replication fork-stalling alkylation lesion, would be of high fidelity. We performed here the first kinetic analyses of E. coli DinB•RecA binary complexes. Whether alone or in a binary complex, DinB inserted the correct deoxyribonucleoside triphosphate (dNTP) opposite either lesion-containing or undamaged template; the incorporation of other dNTPs was largely inefficient. DinB prefers undamaged DNA, but the DinB•RecA binary complex increases its catalytic efficiency on lesion-containing template, perhaps as part of a regulatory mechanism to better respond to alkylation damage. Notably, we find that a DinB derivative with enhanced affinity for RecA, either alone or in a binary complex, is less efficient and has a lower fidelity than DinB or DinB•RecA. This finding contrasts our previous genetic analyses. Therefore, mutagenesis resulting from alkylation lesions is likely limited in cells by the activity of DinB•RecA. These two highly conserved proteins play an important role in maintaining genomic stability when cells are faced with ubiquitous DNA damage. Kinetic analyses are important to gain insights into the mechanism(s) regulating TLS DNA polymerases. Environ. Mol. Mutagen., 2013. © 2013 Wiley Periodicals, Inc.
    Full-text · Article · Mar 2014 · Environmental and Molecular Mutagenesis
  • Source
    • "Among those tested were the cytotoxic 3- methyl adenine (3MeA) lesion that is produced in DNA by the action of S N 2 methylating agents, the major oxidative lesion 8-oxoguanine (8-oxoG), and an abasic site, which can occur spontaneously or as an intermediate of the base excision repair pathway. Tgo-Pol was unable to bypass a stable 3-deaza analog of 3MeA (3dMeA; Plosky et al., 2008) or an abasic site (Figure 5E, lane 2, and data not shown), consistent with these lesions blocking cellular DNA replicases, but could bypass an 8-oxoG, although stalling was visible at the lesion (Figure 5F, lane 2). PPL1 and PPL2 could incorporate a single nucleotide opposite a templated 3dMeA but could not extend further from this terminus (Figure 5E), and both enzymes correctly inserted T opposite the lesion (Figure S6C). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Faithful copying of the genome is essential for life. In eukaryotes, a single archaeo-eukaryotic primase (AEP), DNA primase, is required for the initiation and progression of DNA replication. Here we have identified additional eukaryotic AEP-like proteins with DNA-dependent primase and/or polymerase activity. Uniquely, the genomes of trypanosomatids, a group of kinetoplastid protozoa of significant medical importance, encode two PrimPol-like (PPL) proteins. In the African trypanosome, PPL2 is a nuclear enzyme present in G2 phase cells. Following PPL2 knockdown, a cell-cycle arrest occurs after the bulk of DNA synthesis, the DNA damage response is activated, and cells fail to recover. Consistent with this phenotype, PPL2 replicates damaged DNA templates in vitro, including templates containing the UV-induced pyrimidine-pyrimidone (6-4) photoproduct. Furthermore, PPL2 accumulates at sites of nuclear DNA damage. Taken together, our results indicate an essential role for PPL2 in postreplication tolerance of endogenous DNA damage, thus allowing completion of genome duplication.
    Full-text · Article · Nov 2013 · Molecular Cell
Show more