Human DNA Polymerase ι Incorporates dCTP Opposite Template G via a G.C+ Hoogsteen Base Pair

Nair, D.T.; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

doi:10.1016/j.str.2005.08.010

Cited by 123 publications

(192 citation statements)

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“…The syn conformation of 8-oxo-G presents the Hoogsteen edge exclusively for hydrogen bonding. Polι induces syn conformations on template purines due to an exceptionally narrow active site, which restricts the C1′-C1′dis-tance to under 9 Å (16,17). Accordingly, the C1′-C1′ distance of all four replicating base pairs in the current 8-oxo-G structures is less than 9 Å ( Fig.…”

Section: Resultsmentioning

confidence: 97%

Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota

Kirouac

2011

Proc. Natl. Acad. Sci. U.S.A.

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The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι's biological role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA containing an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equilibrium. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the additional O 8 atom of the lesion base, which influences nucleotide selection, enzymatic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase.DNA replication | Y family polymerase | translesion DNA synthesis | oxidative lesion | DNA mutagenesis

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Section: Resultsmentioning

confidence: 97%

Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota

Kirouac

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…The ternary structures of Pol and Pol with DNA and incoming deoxynucleoside triphosphate have been solved (29,(35)(36)(37)(38)47).…”

Section: Discussionmentioning

confidence: 99%

“…Pol differs strikingly from Pols and and almost all other Pols in that it incorporates nucleotides opposite template purines with much higher efficiency and fidelity than opposite template pyrimidines (6,19,44,49). Moreover, even opposite template purines, Pol exhibits higher catalytic efficiency and fidelity opposite template A than opposite template G. The ternary crystal structures of Pol bound to template A or G and the correct incoming deoxynucleoside triphosphate have shown that the purine template adopts a syn conformation in the Pol active site and forms a Hoogsteen base pair with the incoming nucleotide, which remains in the anti conformation (36)(37)(38). Hoogsteen base pairing explains the basis for the higher efficiency of correct nucleotide incorporation opposite template purines than opposite template pyrimidines, as only the purine bases have the Hoogsteen edge via which they can hydrogen bond with the correct incoming nucleotide.…”

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confidence: 99%

A Role for Yeast and Human Translesion Synthesis DNA Polymerases in Promoting Replication through 3-Methyl Adenine

Johnson

Prakash

et al. 2007

Molecular and Cellular Biology

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3-Methyl adenine (3meA), a minor-groove DNA lesion, presents a strong block to synthesis by replicative DNA polymerases (Pols). To elucidate the means by which replication through this DNA lesion is mediated in eukaryotic cells, here we carry out genetic studies in the yeast Saccharomyces cerevisiae treated with the alkylating agent methyl methanesulfonate. From the studies presented here, we infer that replication through the 3meA lesion in yeast cells can be mediated by the action of three Rad6-Rad18-dependent pathways that include translesion synthesis (TLS) by Pol or -and an Mms2-Ubc13-Rad5-dependent pathway which presumably operates via template switching. We also express human Pols and in yeast cells and show that they too can mediate replication through the 3meA lesion in yeast cells, indicating a high degree of evolutionary conservation of the mechanisms that control TLS in yeast and human cells. We discuss these results in the context of previous observations that have been made for the roles of Pols , , and in promoting replication through the minor-groove N 2 -dG adducts.Replicative DNA polymerase (Pols) are highly sensitive to geometric distortions in DNA; consequently, they are inhibited by the presence of DNA lesions in the template strand. A number of Pols that promote replication through DNA lesions exist in eukaryotes, and they are highly specialized for the roles they play in translesion synthesis (TLS) (40). For example, both yeast and human Pols are highly adept at promoting error-free replication through UV-induced cyclobutane pyrimidine dimers (CPDs) (15,20,50,52), and inactivation of Pol in humans causes the cancer-prone syndrome of the variant form of xeroderma pigmentosum (14,30).Although proficient replication through a DNA lesion such as a CPD can be accomplished by Pol alone, replication through many of the different lesions present in DNA requires the sequential action of two Pols, in which one polymerase inserts the nucleotide opposite the DNA lesion and another polymerase performs the subsequent extension reaction (40,41). Yeast Pol, comprised of the Rev3 catalytic and Rev7 accessory subunits (39), is highly specialized for performing the extension step of TLS (8,19,21,35). Although humans also have the Rev3 and Rev7 proteins, there is no biochemical information available on the role of human Pol in TLS.In addition to Pol, humans have Pols and , which belong to the Y family of Pols (40). Pol differs strikingly from Pols and and almost all other Pols in that it incorporates nucleotides opposite template purines with much higher efficiency and fidelity than opposite template pyrimidines (6,19,44,49). Moreover, even opposite template purines, Pol exhibits higher catalytic efficiency and fidelity opposite template A than opposite template G. The ternary crystal structures of Pol bound to template A or G and the correct incoming deoxynucleoside triphosphate have shown that the purine template adopts a syn conformation in the Pol active site and forms a Hoogsteen base pair with the incom...

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“…It is worthwhile to note, however, that for some polymerases one or more base pairs may be especially sensitive to assay conditions. As an example, consider human Pol ι, which has recently been suggested to protonate N3 of incoming dCTP to facilitate Hoogstein pairing with templating G (30). This protonation event is not requisite for incoming dTTP.…”

Section: Condition Dependence Of Polymerase Fidelitymentioning

confidence: 99%

ASFV DNA Polymerase X Is Extremely Error-Prone under Diverse Assay Conditions and within Multiple DNA Sequence Contexts

2006

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We previously demonstrated that the DNA repair system encoded by the African swine fever virus (ASFV) is both extremely error-prone during the single-nucleotide gap-filling step (catalyzed by ASFV DNA Polymerase X) and extremely error-tolerant during the nick-sealing step (catalyzed by ASFV DNA ligase). On the basis of these findings we have suggested that at least some of the diversity known to exist among ASFV isolates may be a consequence of mutagenic DNA repairwherein damaged nucleotides are replaced with undamaged but incorrect nucleotides by Pol X and the resultant mismatched nicks are sealed by ASFV DNA ligase. Recently, this hypothesis appeared to be discredited by Salas and coworkers [J. Mol. Biol. 2003, 326, 1403-1412 who reported the fidelity of Pol X to be, on average, two orders of magnitude higher than what we previously published. In an effort to address this discrepancy and provide a definitive conclusion about the fidelity of Pol X, herein we examine the fidelity of Pol X-catalyzed single-nucleotide gap-filling in both the steady state and the pre-steady state under a diverse array of assay conditions (varying pH and ionic strength) and within different DNA sequence contexts. These studies corroborate our previously published data (demonstrating the low-fidelity of Pol X to be independent of assay condition/sequence context), do not reproduce the data of Salas et al., and therefore confirm Pol X to be one of the most errorprone polymerases known. These results are discussed in light of ASFV biology and the mutagenic DNA repair hypothesis described above.ASFV 1 causes a disease of varying mortality [depending on the particular isolate (1)] in domestic pigs in Africa, the Iberian Peninsula, and the Caribbean (2). Possessing a large [168-189 kb (3)] double-stranded DNA genome encoding 151 proteins (4), ASFV is one of the most complex viruses known. In its target cells, marcrophages and monocytes (5), ASFV utilizes the host cell nucleus during an early phase of viral DNA synthesis but appears to complete the replication/assembly of its genome in cytoplasmic/perinuclear viral factories (6 -8). Consistent with the latter intracellular location, ASFV encodes its own replicative polymerase in addition to a minimalist DNA repair system consisting of an AP endonuclease (APE), a repair polymerase (Pol X), and an ATP-dependent DNA ligase (4). While this tripartite repair system would appear to have been retained for the purpose of processing spontaneously 2 generated apurinic/apyrimidinic (AP) sites and/or reactive oxygen species (ROS)-induced singlestrand † This work was supported by NIH Grant GM43268. B.J.L. was supported in part by a predoctoral NIH CBIP fellowship (2T32 GM08512).*To whom correspondence should be addressed at the Department of Chemistry [phone: (614) breaks in the viral genome (9), the unique substrate specificities of both Pol X and ASFV DNA ligase have led us to hypothesize that this "repair" system may have subsequently evolved a secondary function, that of viral genome muta...

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Human DNA Polymerase ι Incorporates dCTP Opposite Template G via a G.C+ Hoogsteen Base Pair

Cited by 123 publications

References 44 publications

Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota

Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota

A Role for Yeast and Human Translesion Synthesis DNA Polymerases in Promoting Replication through 3-Methyl Adenine

ASFV DNA Polymerase X Is Extremely Error-Prone under Diverse Assay Conditions and within Multiple DNA Sequence Contexts

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