Incorporation of the Guanosine Triphosphate Analogs 8-Oxo-dGTP and 8-NH2-dGTP by Reverse Transcriptases and Mammalian DNA Polymerases

Abstract

We have measured the efficiencies of utilization of 8-oxo-dGTP and 8-NH 2 -dGTP by human immunodeficiency virus type 1 and murine leukemia virus reverse transcriptases and compared them to those of DNA polymerases ␣ and ␤. Initially, we carried out primer extension reactions in the presence of dGTP or a dGTP analog and the remaining three dNTPs using synthetic DNA and RNA templates. These assays revealed that, in general, 8-NH 2 -dGTP is incorporated and extended more efficiently than 8-oxo-dGTP by all enzymes tested. Second, we determined rate constants for the incorporation of each analog opposite a template cytidine residue using steady state single nucleotide extension kinetics. Our results demonstrated the following. 1) Both reverse transcriptases incorporate the nucleotide analogs; discrimination against their incorporation is a function primarily of K m or V max depending on the analog and the enzyme. 2) Discrimination against the analogs is more stringent with the DNA template than with a homologous RNA template. 3) Polymerase ␣ exhibits a mixed kinetic phenotype, with a large discrimination against 8-oxo-dGTP but a comparatively higher preference for 8-NH 2 -dGTP. 4) Polymerase ␤ incorporates both analogs efficiently; there is no discrimination with respect to K m and a significantly lower discrimination with respect to V max when compared with the other polymerases.Cellular DNA is subject to modifications by endogenous processes as well as from exposure to exogenous agents. If unrepaired, alterations of the nucleoside residues in DNA can result in misincorporations during DNA replication. Base damage can also occur at the level of the deoxynucleoside and/or deoxynucleotide (1, 2). For these to become mutagenic they first need to be incorporated into DNA at the time of DNA synthesis. In this paper, we have investigated the in vitro utilization of two potentially mutagenic analogs of dGTP, 8-oxo-dGTP and 8-NH 2 -dGTP, by viral and mammalian DNA polymerases that lack 3Ј 3 5Ј exonucleolytic activity.7,8-Dihydro-8-oxodeoxyguanosine, also referred to as 8-hydroxydeoxyguanosine (8-oxo-dG), 1 in cellular DNA is a byproduct of oxygen damage. Reactive oxygen species such as singlet O 2 , hydrogen peroxide (H 2 O 2 ), hydroxyl (OH ⅐ ), and superoxide (O 2 . ) radicals, generated from the actions of ionizing radiation, chemical mutagens, and endogenous processes, are believed to be responsible for this damage. In particular, hydroxyl radicals are implicated in reactions at the C-8 position of 2Ј-deoxyguanosine in DNA to produce the lesion 8-oxo-dG (3, 4). Alternatively, the lesion is generated at the level of the nucleoside triphosphate, where dGTP is converted to 8-oxo-dGTP by reactive oxygen species and subsequently incorporated into DNA. Irrespective of how it is generated, the lesion in DNA is mutagenic. DNA polymerases can insert nucleotides opposite the lesion and synthesize past it (5-7). Although 8-oxo-dG can theoretically form base pairs with each of the four deoxynucleosides, most polymerases studied i...

Enhanced expression and activity of DNA polymerase β in human ovarian tumor cells: impact on sensitivity towards antitumor agents

“…One of the main characteristics of Pol b is its low capacity to discriminate between nucleotide analogs (Bouayadi et al, 1997;Kamath-Loeb et al, 1997). We hypothesized that cells up-regulating Pol b may display hypersensitivity to chemotherapeutic drugs which are nucleotides analogs such as 6-TG.…”

“…Pol b can be distinguished from the polymerases involved in the genome replication by its high in®delity in replicating DNA, due to the lack of associated proofreading activity (Kornberg and Baker, 1992), a poor ability to discriminate oxidized nucleotides (Kamath-Loeb et al, 1997), and an ability to e ciently incorporate into DNA nucleotide analogs such as ddCTP (Copeland et al, 1992), AZT-TP (Parker et al, 1991), as well as ara-CTP (Ohno et al, 1988). Even during the single nucleotide gap ®lling step of the BER pathway, Pol b has been shown to be errorprone (Oshero et al, 1999).…”

Effects of base excision repair gene polymorphisms on pancreatic cancer survival

“…20 The primary function of POLB is thought to be in the processing of damaged bases via the BER pathway, thereby protecting cells from DNA damage-induced cytotoxicity. 21,22 At the transcriptional level, POLB is overexpressed in many cancer cells. 23 Overexpression of POLB has been associated with an increased chromosome instability and tumorigenesis as well as with resistance to many DNA-damaging agents, including chemotherapeutic drugs.…”

Deregulated DNA polymerase β strengthens ionizing radiation-induced nucleotidic and chromosomal instabilities

“…In the course of the identi®cation of molecular factors responsible for the acquisition of a mutator phenotype by deregulation of expression of an error-prone DNA polymerase, we showed the ability of excess Pol b to enhance incorporation of mutagenic nucleotide analogs produced by IR. This is not surprising regarding the features that distinguish pol b from the replicative polymerases: the lack of associated proofreading activity, which results in low ®delity in replicating DNA in vitro, and a poor ability to discriminate nucleotides at the level of binding (Copeland et al, 1992;Kamath-Loeb et al, 1997). Evidence that a mutator phenotype can result from nucleotide instability was delineated by studies on microsatellite instability in hereditary non-polyposis colon cancer cells (HNPCC) (Ionov et al, 1993;Peinado et al, 1992), which present de®ciencies in genes involved in DNA mismatch repair, resulting in mutations at the nucleotidic level that were well identi®ed at repetitive nucleotide sequences that undergo expansion or retraction between or within genes (Markowitz et al, 1995;Rampino et al, 1997).…”