Lesion (in)tolerance reveals insights into DNA replication fidelity

Freisinger, Eva; Grollman, Arthur P.; Miller, Holly; Kisker, Caroline

doi:10.1038/sj.emboj.7600158

Cited by 123 publications

(229 citation statements)

References 48 publications

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“…Accommodation of lesion-induced template distortions by high-fidelity polymerases has also been observed for other types of DNA lesions that are less bulky than G-AF or G-AAF but also impede DNA replication. Structures of 8ox-oguanine, cyclobutane pyrimidine dimers, or abasic lesions bound by T7 DNA polymerase (67,68), RB69 polymerase (69,70), or BF (62) reveal that template distortions induced by these lesions do not necessarily result in distortions to the polymerase active site. However, interactions between the lesioned duplex and the polymerase are lost, thereby impairing lesion bypass and promoting partitioning of the DNA duplex to the exonuclease domain in the case of RB69 (69,70).…”

Section: Anti Conformation Of G-af Promotes Accurate Replication-mentioning

confidence: 99%

“…Structures of 8ox-oguanine, cyclobutane pyrimidine dimers, or abasic lesions bound by T7 DNA polymerase (67,68), RB69 polymerase (69,70), or BF (62) reveal that template distortions induced by these lesions do not necessarily result in distortions to the polymerase active site. However, interactions between the lesioned duplex and the polymerase are lost, thereby impairing lesion bypass and promoting partitioning of the DNA duplex to the exonuclease domain in the case of RB69 (69,70). Rather than bypass lesions using mechanisms of replication for unmodified DNA, high-fidelity polymerases employ non-templated incorporation of dATP (the "A" rule; Ref.…”

Section: Anti Conformation Of G-af Promotes Accurate Replication-mentioning

confidence: 99%

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Observing Translesion Synthesis of an Aromatic Amine DNA Adduct by a High-fidelity DNA Polymerase

Hsu

Kiefer

Burnouf³

et al. 2004

Journal of Biological Chemistry

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Aromatic amines have been studied for more than a half-century as model carcinogens representing a class of chemicals that form bulky adducts to the C8 position of guanine in DNA. Among these guanine adducts, the N-(2-deoxyguanosin-8-yl)-aminofluorene (G-AF) and N-2-(2-deoxyguanosin-8-yl)-acetylaminofluorene (G-AAF) derivatives are the best studied. Although G-AF and G-AAF differ by only an acetyl group, they exert different effects on DNA replication by replicative and high-fidelity DNA polymerases. Translesion synthesis of G-AF is achieved with high-fidelity polymerases, whereas replication of G-AAF requires specialized bypass polymerases. Here we have presented structures of G-AF as it undergoes one round of accurate replication by a high-fidelity DNA polymerase. Nucleotide incorporation opposite G-AF is achieved in solution and in the crystal, revealing how the polymerase accommodates and replicates past G-AF, but not G-AAF. Like an unmodified guanine, G-AF adopts a conformation that allows it to form Watson-Crick hydrogen bonds with an opposing cytosine that results in protrusion of the bulky fluorene moiety into the major groove. Although incorporation opposite G-AF is observed, the C:G-AF base pair induces distortions to the polymerase active site that slow translesion synthesis.

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Section: Anti Conformation Of G-af Promotes Accurate Replication-mentioning

confidence: 99%

Section: Anti Conformation Of G-af Promotes Accurate Replication-mentioning

confidence: 99%

Observing Translesion Synthesis of an Aromatic Amine DNA Adduct by a High-fidelity DNA Polymerase

Hsu

Kiefer

Burnouf³

et al. 2004

Journal of Biological Chemistry

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“…The rear wall of this binding pocket, which includes residues L561, N564 and S565 as shown in Fig. 3 of (12), is presumed to reinforce the coplanar arrangement of the nascent base pair (12). According to the ternary structure of RB69 pol (29), the side chain of L561 is an obvious protrusion into the nucleotide binding pocket that may clash with a bulge on the major groove side of a mispair.…”

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confidence: 99%

The L561A Substitution in the Nascent Base-Pair Binding Pocket of RB69 DNA Polymerase Reduces Base Discrimination

et al. 2006

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Several variants of RB69 DNA polymerase (RB69 pol) with single-site replacements in the nascent base-pair binding pocket are less discriminating with respect to non-complementary dNMP incorporation than the wild-type enzyme. To quantify the loss in base selectivity, we determined the transient-state kinetic parameters for incorporation of correct and all combinations of incorrect dNMPs by the exonuclease deficient form of one of these RB69 pol variants, L561A, using rapid chemical quench assays. The L561A variant did not significantly alter the k pol and K D values for incorporation of correct dNMPs, but it showed increased incorporation efficiency (k pol / K D ) for mispaired bases relative to the wild type enzyme. The incorporation efficiency for mispaired bases by the L561A variant ranged from 1.5 × 10 −5 µM −1 s −1 for dCMP opposite templating C to 2 × 10 −3 µM −1 s −1 for dAMP opposite templating C. These k pol /K D values are 3-60 fold greater than those observed with the wild type enzyme. The effect of the L561A replacement on the mutation frequency in vivo was determined by infecting E. coli, harboring a plasmid encoding the L561A variant of RB69 pol, with T4 phage bearing a mutant rII locus and the rates of reversions to rII + were scored. The exonuclease-proficient RB69 pol L561A displayed a weak mutator phenotype. In contrast, no progeny phage were produced after infection of E. coli, expressing an exonuclease-deficient RB69 pol L561A, with either mutant or wild type T4 phage. This dominant-lethal phenotype was attributed to error catastrophe caused by the high rate of mutation expected from combining the pol L561A and exo − mutator activities. Keywords base selectivity; transient-state kinetic parameters; mutation frequency; modeling of mismatches DNA polymerase is the central component of replicases that are responsible for faithfully copying DNA. Despite the fact that all four dNTPs are potential substrates, replicative DNA polymerases are able to limit the incorporation of mismatched bases to about one in a million [for reviews see (1-8)]. Although this error frequency is acceptable for phage replication, it is still far too high to maintain genetic integrity during cell proliferation in † Supported by National Institutes of Health, Public Health Service grants GM63276 (to W.K.) and TW006626 (to A.B.). J.W.D. was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences. * To whom correspondence and reprint requests should be addressed. telephone, (203) 785-4599; fax, (203) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript more complex organisms. To increase fidelity, cells have a number of mechanisms for correcting errors, including the ability of the replicative DNA polymerase itself, or an associated subunit, to excise misincorporated bases. In addition, there are specialized DNA polymerases that confront DNA damage inflicted by radiation, oxidation, alkylating agents, etc. which block the progress of replic...

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“…S1) have shown that purines, in particular adenosine, and to a lesser extent guanosine, are most frequently incorporated opposite the lesion. The strong preference for adenosine has been termed "A-rule" (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).…”

mentioning

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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Lesion (in)tolerance reveals insights into DNA replication fidelity

Cited by 123 publications

References 48 publications

Observing Translesion Synthesis of an Aromatic Amine DNA Adduct by a High-fidelity DNA Polymerase

Observing Translesion Synthesis of an Aromatic Amine DNA Adduct by a High-fidelity DNA Polymerase

The L561A Substitution in the Nascent Base-Pair Binding Pocket of RB69 DNA Polymerase Reduces Base Discrimination

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

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