Mechanism of Abasic Lesion Bypass Catalyzed by a Y-family DNA Polymerase

Fiala, Kevin A.; Hypes, Cameron; Suo, Zucai

doi:10.1074/jbc.m610718200

Cited by 60 publications

(121 citation statements)

References 42 publications

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“…Running Start Assay-The running start assay was performed as previously described (17,18,26). Briefly, a preincubated solution of 5Ј-32 P-labeled DNA (100 nM) and Dpo4 (100 nM) in buffer R was rapidly mixed with a solution containing all four dNTPs (200 M each) at 37°C via a rapid chemical-quench flow apparatus (KinTek).…”

Section: Methodsmentioning

confidence: 99%

“…Singleturnover dNTP incorporation assays were employed to obtain the k p and K d,dNTP as previously described (14,15,17,26). Briefly, a preincubated solution of Dpo4 (120 nM) and 5Ј-32 Plabeled DNA (30 nM) in buffer R was mixed with increasing concentrations of a dNTP.…”

Section: Methodsmentioning

confidence: 99%

“…Second, S. solfataricus would have to encode the necessary enzymes like nitro reductase to metabolize 1-NP and form dG AP (38). We chose to study dG AP bypass catalyzed by Dpo4 because (i) the kinetic mechanism of nucleotide incorporation into undamaged DNA catalyzed by Dpo4 has been previously elucidated by our laboratory (14), (ii) there are many published crystal structures of the Dpo4 ternary complexes which contain various DNA lesions (16, 20, 22, 25, 27, 28, 40 -43), (iii) Dpo4 is the only Y-family DNA polymerase encoded by S. solfataricus and, thus, is responsible for most translesion synthesis events in that organism (38), and (iv) we have established kinetic mechanisms and pathways for the bypass of an abasic site (17) and a cisplatin-d(GpG) adduct (26) catalyzed by Dpo4. In addition, both Dpo4 and eukaryotic DNA polymerase are DinB (damageinduced protein) homologs (45,46), and the bypass abilities for a spectrum of DNA lesions are similar to those of eukaryotic DNA polymerase (24,47,48).…”

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confidence: 99%

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Mechanistic Studies of the Bypass of a Bulky Single-base Lesion Catalyzed by a Y-family DNA Polymerase

Sherrer

Brown

Pack

et al. 2009

Journal of Biological Chemistry

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104

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1-Nitropyrene, the most abundant nitro polycyclic aromatic hydrocarbon in diesel emissions, has been found to react with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dG AP ). This bulky adduct has been shown to induce genetic mutations, which may implicate Y-family DNA polymerases in its bypass in vivo. To establish a kinetic mechanism for the bypass of such a prototype single-base lesion, we employed pre-steady-state kinetic methods to investigate individual nucleotide incorporations upstream, opposite, and downstream from a site-specifically placed dG AP lesion catalyzed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. Dpo4 was able to bypass dG AP but paused strongly at two sites: opposite the lesion and immediately downstream from the lesion. Both nucleotide incorporation efficiency and fidelity decreased significantly at the pause sites, especially during extension of the bypass product. Interestingly, a 4-fold tighter binding affinity of damaged DNA to Dpo4 promoted catalysis through putative interactions between the active site residues of Dpo4 and 1-aminopyrene moiety at the first pause site. In the presence of a DNA trap, the kinetics of nucleotide incorporation at these sites was biphasic in which a small, fast phase preceded a larger, slow phase. In contrast, only a large, fast phase was observed during nucleotide incorporation at non-pause sites. Our kinetic studies support a general kinetic mechanism for lesion bypass catalyzed by numerous DNA polymerases.Environmental pollutants have been shown to impact human health at the molecular level. One detrimental route is the modification of genomic DNA and nucleotides (1). If DNA lesions are not recognized and removed by the cellular DNA repair machinery, they will stall replicative DNA polymerases (2-8).To rescue DNA replication, cells employ lesion bypass DNA polymerases to traverse unrepaired lesions. Most of these enzymes belong to the Y-family of DNA polymerases. The Y-family enzymes possess relatively flexible and solvent-accessible active sites to accommodate bulky DNA lesions (9, 10). However, Y-family DNA polymerases catalyze DNA synthesis over undamaged DNA with low fidelity and poor processivity (6, 10 -12). The Y-family DNA polymerases have been identified in all three domains of life, e.g. four in humans (DNA polymerases , , , and Rev1), two in Escherichia coli (DNA polymerases IV and V), and one in Sulfolobus solfataricus (Dpo4). Because Dpo4 can be expressed in E. coli and purified with a high yield, it has been extensively studied in vitro as a prototype Y-family enzyme. Dpo4 catalyzes DNA synthesis on an undamaged DNA template with a fidelity of one error per 1,000 -10,000 nucleotide incorporations based on presteady-state kinetic analysis from 37 to 56°C (13-15). Dpo4 is capable of bypassing a myriad of DNA lesions including apurinic/apyrimidinic (abasic) sites (16 -19), 8-oxo-7,8-dihydro-2Ј-deoxyguanosine (20, 21), 1,N 2 -etheno(⑀)guanosine (22), cis-syn thymine-thymine dimer (23-...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Mechanistic Studies of the Bypass of a Bulky Single-base Lesion Catalyzed by a Y-family DNA Polymerase

Sherrer

Brown

Pack

et al. 2009

Journal of Biological Chemistry

Self Cite

104

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“…Most translesion DNA polymerases follow various loop-out mechanisms (11,(21)(22)(23)(24)(25). Thereby, the nucleotide selection is influenced by the following upstream templating bases resulting in deletions and complex mutation spectra.…”

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confidence: 99%

Amino Acid Templating Mechanisms in Selection of Nucleotides Opposite Abasic Sites by a Family A DNA Polymerase

Obeid

Welte

Diederichs

et al. 2012

Journal of Biological Chemistry

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Background: Abasic sites are the most frequent DNA lesions and are often bypassed by incorporating an adenosine opposite that lesion. Results: We determined structures of DNA polymerase in complex with different nucleotides opposite an abasic site. Conclusion: Interaction of the incoming nucleotide with a single amino acid governs nucleotide selection opposite abasic sites. Significance: This work furthers the understanding of the bypass of a mutagenic lesion by DNA polymerases.

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“…DNA polymerases of these organisms stall specifically 4 nucleotides (nt) ahead of template dU, but assays in vitro indicate that this lesion is eventually bypassed with insertion of dA (40,41). Alternatively, if dU becomes excised by a corresponding DNA N-glycosylase but is not repaired before replication, dA is likely to be inserted opposite the resulting noninstructional abasic site, based on assays performed in vitro (42,43) and in vivo (D. W. Grogan and C. J. Sakofsky, unpublished data). Accordingly, either route leads to a G·C-to-A·T transition.…”

Section: An Archaeal Model Of Conditional Endogenous Mutagenesismentioning

confidence: 99%

Endogenous Mutagenesis in Recombinant Sulfolobus Plasmids

Sakofsky

Grogan

2013

J Bacteriol

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Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the ␤-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac ؊ mutants also grew faster than the Lac ؉ parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 ؋ 10 ؊4 mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G·C-to-A·T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.T he universal need of organisms to replicate genomes accurately, and in coordination with cell division, has special significance for "extreme" or "hyper-" thermophiles, which grow optimally at temperatures that kill mesophilic organisms and denature their macromolecules (1). The hyperthermophilic archaea which colonize terrestrial and marine hydrothermal systems represent diverse and ancient lineages, and all grow optimally at temperatures that accelerate DNA decomposition reactions by orders of magnitude relative to those in mesophiles (2). The idea that these archaea should have effective strategies of genome protection which compensate for diverse damaging effects of high temperature thus seems logical and is supported by the low rates of replication errors measured in several Sulfolobus isolates (3, 4). The extreme phylogenetic divergence separating archaea from bacteria and eukaryotes (5) further suggests that the strategies which preserve genome integrity in hyperthermophilic archaea may include molecular features not identified in model organisms. This idea remains consistent with the unusual gene inventories and certain biochemical features of DNA metabolism in these archaea (6). The mechanisms that maintain genomic integrity in hyperthermophilic archaea remain challenging to identify in vivo, however. Recent studies, for example, found that several putative ...

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Mechanism of Abasic Lesion Bypass Catalyzed by a Y-family DNA Polymerase

Cited by 60 publications

References 42 publications

Mechanistic Studies of the Bypass of a Bulky Single-base Lesion Catalyzed by a Y-family DNA Polymerase

Mechanistic Studies of the Bypass of a Bulky Single-base Lesion Catalyzed by a Y-family DNA Polymerase

Amino Acid Templating Mechanisms in Selection of Nucleotides Opposite Abasic Sites by a Family A DNA Polymerase

Endogenous Mutagenesis in Recombinant Sulfolobus Plasmids

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