Article

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: 8.81). 05/2008; 36(7):2152-62. DOI: 10.1093/nar/gkn058
Source: PubMed

ABSTRACT N3-methyl-adenine (3MeA) is the major cytotoxic lesion formed in DNA by S(N)2 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 alpha and delta. In contrast, human polymerases eta, iota and kappa, as well as Saccharomyces cerevisiae poleta 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, poleta (Rad30) contributes to the survival of cells exposed to methyl methanesulfonate (MMS) and in the absence of Mag1, Rad30 and Rev3, human polymerases eta, iota and kappa are capable of restoring MMS-resistance to the normally MMS-sensitive strain.

0 Bookmarks
 · 
111 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The catalytic effects of C8-H···O5′ hydrogen bond on 3-methyl-2′-deoxyadenosine (3-MDA) depurination has been studied based on the properties of substituents located at the O5′ position using the quantum mechanical calculations at the B3LYP/6-311++G(d,p) level of theory. The energy of intramolecular hydrogen bond (E HB) was estimated based on the topological properties of the hydrogen bond critical point. A linear correlation was found between the E HB and the N-glycosidic bond length. The ED/EW substituents enlarge/contract the bond and facilitate/hinder depurination process via the intramolecular interaction. The changes in the charge distributions on the sugar ring and nucleobase, induced by the C8-H···O5′ hydrogen bond, are in good relationship with the C1′-O4′ and N-glycosidic bond length. The intramolecular interaction enhances the N7 proton affinity, which its protonation increases positive charge on the sugar ring and facilitates the depurination process.
    Structural Chemistry 04/2014; 26(2). DOI:10.1007/s11224-014-0493-4 · 1.90 Impact Factor
  • Source
    [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.
    Analytical Biochemistry 03/2015; DOI:10.1016/j.ab.2015.03.002 · 2.31 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (Pcna(K164R)) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
    Nucleic Acids Research 12/2014; DOI:10.1093/nar/gku1301 · 8.81 Impact Factor

Preview

Download
2 Downloads
Available from