Translesional Synthesis on DNA Templates Containing a Single Abasic Site

Abstract

Site-specifically modified oligodeoxynucleotides containing a single natural abasic site or a chemically synthesized (tetrahydrofuran or deoxyribitol) model abasic site were used as templates for primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I or by calf thymus DNA polymerase ␣. Analysis of the fully extended products of these reactions indicated that both polymerases preferentially incorporate dAMP opposite the natural abasic site and tetrahydrofuran, while DNA templates containing the ring-opened deoxyribitol moiety block translesional synthesis, promoting sequence context-dependent deletions. The frequency of nucleotide insertion opposite the three types of abasic sites follows the order dAMP > dGMP > dCMP > dTMP. The frequency of chain extension was highest when dAMP was positioned opposite a natural abasic site. The frequency of translesional synthesis past abasic sites follows the order tetrahydrofuran > deoxyribose > deoxyribitol. The Klenow fragment promotes blunt end addition of dAMP; this reaction was much less efficient than insertion of dAMP opposite an abasic site. We conclude that the miscoding potential of a natural abasic site in vitro closely resembles that of its tetrahydrofuran analog. Ring-opened abasic sites favor deletions. Studies with polymerase ␣ in vitro predict preferential incorporation of dAMP at abasic sites in mammalian cells.Abasic sites in DNA arise spontaneously by hydrolysis, a process that can be accelerated by modification of purine bases (1) and by the catalytic action of N-glycosylases that remove damaged bases from DNA (2-4). The natural abasic site (Ab) 1 exists as an equilibrium mixture of the cyclic hemiacetal and open chain aldehyde forms of 2Ј-deoxyribose (see Fig. 1) and is subject to ␤-elimination. This chemical reaction leads to strand scission (5); for that reason, structural analogs of deoxyribose have often been used to explore biological properties of abasic sites in DNA (6 -10). Abasic site analogs include deoxyribitol, a model for the open chain form of the sugar (6, 7), and tetrahydrofuran, an isosteric and isoelectronic analog of deoxyribose (8, 9) that is cleaved by type II AP endonucleases, but is not subject to ␤-elimination (9).The miscoding properties of natural and synthetic abasic sites have been investigated under a variety of experimental conditions using randomly or site-specifically modified DNA (6,7,(11)(12)(13)(14)(15). The relative frequency of base incorporation opposite abasic sites and of chain extension from the 3Ј-primer terminus has also been reported (8, 9). While natural and synthetic abasic sites are structurally similar, their mutagenic potential has not been compared under the same experimental conditions.In Escherichia coli, synthesis past abasic sites in vitro (6 -9, 11-13) and in vivo (14, 15) is accompanied by preferential incorporation of dAMP opposite the lesion, a phenomenon known as the "A rule" (16). In eukaryotes, the presence of abasic sites in DNA is associated with differ...

DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency

“…Bypass of 8-oxodA, 8-oxodG, and abasic lesions by several DNA polymerases has been extensively characterized [4,[10][11][12]15,26]. In addition to impacting the rates of nucleotide insertion and extension, these modified bases can pair with normal bases and increase the rate of mutagenesis.…”

“…We studied the mutagenic potential of 8-oxodA, 8-oxodG, and abasic sites by sequencing PCR products derived from templates with these modified bases. In translesional syntheses catalyzed by the Klenow fragment or DNA polymerase α, abasic lesions (either deoxyribitol or tetrahydrofuran) preferentially instruct the insertion of dAMP [15,26]. In addition, 1 and 2-bp deletions have been detected opposite abasic sites where the frequency of deletion formation is dependent on the sequence context at the site of modification [15,26].…”

“…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].…”