Effects of a Guanine-derived Formamidopyrimidine Lesion on DNA Replication

Abstract

2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/ apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3-5 exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (f ins ‫؍‬ V max /K m for insertion) and extension (f ext ‫؍‬ V max /K m for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative f ins ‫؍‬ 1) > > dTMP (2.4 ؋ 10 ؊4 ) Ϸ dAMP (8.1 ؋ 10 ؊5 ) > dGMP (4.5 ؋ 10 ؊7 )), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative f ext ‫؍‬ 1) > mFapyG:T (4.6 ؋ 10 ؊3 ) > > mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.Reactive oxygen species formed by aerobic metabolism, phagocytic blood cells upon inflammation, ionizing radiation, and photosensitized reactions generate structurally diverse oxidative damage to DNA that stores vital genetic information of cells (1-3). Base lesions thus formed are generally restored by the base excision repair pathway involving multiple enzymes such as N-glycosylase/AP 1 lyase, AP endonuclease, DNA polymerase, and DNA ligase (4, 5). However, if left unrepaired, they result in mutations and/or cell death. It has also been implied that oxidative DNA damage is involved in carcinogenesis and various degenerative diseases (6, 7). 7,8-Dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) have been identified as major products in the reaction of DNA with reactive oxygen species (Fig. 1A) (1, 2). When formed in DNA, 8-oxoG in a template directs incorporation of non-cognate dAMP as well as cognate dCMP during translesion synthesis by DNA polymerases, thereby inducing GC-to-TA transversions (8, 9). Similarly, when formed in a cellular nucleotide pool, a 2Ј-deoxyribonucleotide form of 8-oxoG can be incorporated opposite adenine as well as cytosine in a DNA template, inducing AT-to-CG transversions (10 -12). Mispair formation between 8-oxoG and adenine leading to mutation involves an unusual syn-conformer of 8-oxoG that can form two hydrogen bonds between O-6 and N-7-H in 8-oxoG and N-6 -H and N-1 in adenine without introducing significan...

DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency

“…Replication across these lesions by DNA polymerases results in the misincorporation of nucleotides and the establishment of mutations in daughter chromosomes [1][2][3][4][5]. The synthesis of oligonucleotides containing chemically defined bases permits the study of translesion synthesis and consequent misincorporation by a number of DNA polymerases [6][7][8][9][10][11][12][13][14][15][16]. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) can assume either a syn or anti-orientation depending on its base pairing partner [6][7][8][9].…”

“…When nucleotides are presented individually, these two enzymes insert dTMP and to a lesser extent dGMP which demonstrates that 8-oxodA has limited mutagenic potential [12]. The Klenow fragment and DNA polymerase α preferentially insert dAMP opposite both synthetic and natural abasic sites [14][15][16]. Abasic sites also promote sequence-dependent 1 and 2 bp deletions [14][15][16].…”

“…The Klenow fragment and DNA polymerase α preferentially insert dAMP opposite both synthetic and natural abasic sites [14][15][16]. Abasic sites also promote sequence-dependent 1 and 2 bp deletions [14][15][16]. In addition to miscoding, misorientation of the 3' hydroxyl of mismatched bases at the primer terminus can stall polymerization by DNA polymerase I [1,10].…”

Repair of Formamidopyrimidines in DNA Involves Different Glycosylases

“…Experiments using the methylated analogue of FapyG, i.e. 2,6-diamino-4-hydroxy-5-Nmethylformamidopyrimidine (Me-FapyG), have suggested that formamidopyrimidines might also constitute blocks to DNA polymerases (10,11). It is noteworthy, however, that FapyG and FapyA are chemically distinct from Me-FapyG.…”

Methylated DNA-binding domain 1 and methylpurine–DNA glycosylase link transcriptional repression and DNA repair in chromatin

“…We first prepared four 30-base paired oligodeoxynucleotides containing CG ϫ CG, mCG ϫ mCG, mCG ϫ mCmG, or CG ϫ CmG in a unique position by using a primer extension reaction as described (Fig. 5A) (40,41). An electrophoretic mobility-shift assay was performed by using these double-stranded DNAs and bacterially expressed MBD of MBD1 (Fig.…”