Human DNA Polymerase κ Encircles DNA: Implications for Mismatch Extension and Lesion Bypass

Lone, Samer; Townson, Sharon A.; Uljon, Sacha N.; Johnson, Robert E.; Brahma, Amrita; Nair, D.T.; Prakash, Satya; Prakash, Louise; Aggarwal, Aneel K.

doi:10.1016/j.molcel.2007.01.018

Cited by 211 publications

(387 citation statements)

References 47 publications

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“…To account for this observation, a misalignment mechanism whereby G* is looped out, and the 5Ј-neighboring G serves as a template for C insertion was suggested (36). Structural superposition of human Y-family pol (37) and Dpo4 indicates that pol may use the same additional binding site to harbor the looped-out adduct, which cannot fit into the active site without intercalation. Pol could stabilize the adducted base better than Dpo4 by stacking the G base with Phe 171, which is equivalent to Lys 78 in Dpo4 (SI Fig.…”

Section: Bp-dg Looping-out and ؊1 Frameshift Mutationsmentioning

confidence: 99%

See 1 more Smart Citation

A structural gap in Dpo4 supports mutagenic bypass of a major benzo[ a ]pyrene dG adduct in DNA through template misalignment

Bauer

Xing²,

Yagi³

et al. 2007

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

139

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Erroneous replication of lesions in DNA by DNA polymerases leads to elevated mutagenesis. To understand the molecular basis of DNA damage-induced mutagenesis, we have determined the x-ray structures of the Y-family polymerase, Dpo4, in complex with a DNA substrate containing a bulky DNA lesion and incoming nucleotides. The DNA lesion is derived from an environmentally widespread carcinogenic polycyclic aromatic hydrocarbon, benzo[ a ]pyrene (BP). The potent carcinogen BP is metabolized to diol epoxides that form covalent adducts with cellular DNA. In the present study, the major BP diol epoxide adduct in DNA, BP- N 2 -deoxyguanosine (BP–dG), was placed at a template–primer junction. Three ternary complexes reveal replication blockage, extension past a mismatched lesion, and a −1 frameshift mutation. In the productive structures, the bulky adduct is flipped/looped out of the DNA helix into a structural gap between the little finger and core domains. Sequestering of the hydrophobic BP adduct in this new substrate-binding site permits the DNA to exhibit normal geometry for primer extension. Extrusion of the lesion by template misalignment allows the base 5′ to the adduct to serve as the template, resulting in a −1 frameshift. Subsequent strand realignment produces a mismatched base opposite the lesion. These structural observations, in combination with replication and mutagenesis data, suggest a model in which the additional substrate-binding site stabilizes the extrahelical nucleotide for lesion bypass and generation of base substitutions and −1 frameshift mutations.

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Section: Bp-dg Looping-out and ؊1 Frameshift Mutationsmentioning

confidence: 99%

“…5C). Pol also has a unique ''N-clasp'' to encircle the DNA for tight DNA binding (37). These two structural features of pol may contribute to its efficient bypass of the BP adduct.…”

Section: Bp-dg Looping-out and ؊1 Frameshift Mutationsmentioning

confidence: 99%

A structural gap in Dpo4 supports mutagenic bypass of a major benzo[ a ]pyrene dG adduct in DNA through template misalignment

Bauer

Xing²,

Yagi³

et al. 2007

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

139

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“…This N-clasp has been shown to encircle a normal primer/template pair and connects and fastens the flexible LF domain to the catalytic core (20). Human Polκ (hPolκ) with a 90-residue (residues 91-559) or a 67-residue truncated N-clasp (residues 68-526) has diminished catalytic activity compared with the native catalytic domain (residues 1-526) or the domain without the first 18 residues (residues 19-526) (20,22).…”

Section: Significancementioning

confidence: 99%

“…The looped out bulky minor groove adduct is accommodated in a structural gap between the catalytic core (composed of palm, finger, and thumb domains) and the little finger (LF) domain. A ternary complex crystal structure of human Polκ with native DNA has been reported (20). As the DNA adduct is absent in the Polκ structure, it is not known how a BPDE-dG lesion is accommodated and how dCTP is selected by Polκ.…”

mentioning

confidence: 99%

Variants of mouse DNA polymerase κ reveal a mechanism of efficient and accurate translesion synthesis past a benzo[ a ]pyrene dG adduct

Liu

Yang

Tang

et al. 2014

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

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DNA polymerase κ (Polκ) is the only known Y-family DNA polymerase that bypasses the 10S (+)-trans-anti-benzo[a]pyrene diol epoxide (BPDE)-N 2 -deoxyguanine adducts efficiently and accurately. The unique features of Polκ, a large structure gap between the catalytic core and little finger domain and a 90-residue addition at the N terminus known as the N-clasp, may give rise to its special translesion capability. We designed and constructed two mouse Polκ variants, which have reduced gap size on both sides [Polκ Gap Mutant (PGM) 1] or one side flanking the template base (PGM2). These Polκ variants are nearly as efficient as WT in normal DNA synthesis, albeit with reduced accuracy. However, PGM1 is strongly blocked by the 10S (+)-trans-anti-BPDE-N 2 -dG lesion. Steady-state kinetic measurements reveal a significant reduction in efficiency of dCTP incorporation opposite the lesion by PGM1 and a moderate reduction by PGM2. Consistently, Polκ-deficient cells stably complemented with PGM1 GFP-Polκ remained hypersensitive to BPDE treatment, and complementation with WT or PGM2 GFP-Polκ restored BPDE resistance. Furthermore, deletion of the first 51 residues of the N-clasp in mouse Polκ (mPolκ 52-516 ) leads to reduced polymerization activity, and the mutant PGM2 52-516 but not PGM1 52-516 can partially compensate the N-terminal deletion and restore the catalytic activity on normal DNA. However, neither WT nor PGM2 mPolκ 52-516 retains BPDE bypass activity. We conclude that the structural gap physically accommodates the bulky aromatic adduct and the N-clasp is essential for the structural integrity and flexibility of Polκ during translesion synthesis.translesion DNA synthesis | polycyclic aromatic hydrocarbons B enzo[a]pyrene (BP) is one of the best characterized polycyclic aromatic hydrocarbons generated during incomplete fuel combustion, in tobacco smoke, and in cooked food. The most mutagenic and tumorigenic metabolite of BP in vivo is the (+)-7R,8S,9S,10R-BP dihydrodiol epoxide [(+)-anti-BPDE] (1), which reacts readily with the exocyclic amino groups of guanine residues in DNA to form the major 10S (+)-trans-anti-BPDE-N 2 -dG adduct (2) (abbreviated as BPDE-dG hereafter). This adduct typically impedes normal DNA replication. Translesion DNA synthesis (TLS), which provides one mode of DNA damage tolerance, uses specialized bypass polymerases to synthesize DNA opposite and beyond a variety of replication-blocking lesions, thus avoiding replication fork collapse (3). The Y-family DNA polymerases are specialized for TLS. To date, four of them, namely DNA polymerase (Pol) η, Polι, Polκ, and REV1, have been identified in mammals (4-6). Polκ is the only one with homologs in bacteria (DinB; also known as Pol IV in Escherichia coli), archaea [DNA polymerase IV (Dpo4) in Sulfolobus solfataricus and DinB homologue (Dbh) in Sulfolobus acidocaldarius] and all eukaryotes. The mouse and human POLK gene are expressed ubiquitously, with highest levels in testis (7).Human Polκ can accurately incorporate dCMP opposite the BPDE-dG adduct ...

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“…Of particular interest are the human lesion-bypass DNA polymerases η, ι, κ and Rev 1. Crystal structures of these enzymes from humans [46,47] and yeast [48,49] have been obtained, as has a lesion-containing yeast Pol η structure [50]. Complemented by biochemical studies [3,50], these structures reveal unique features for each enzyme that reflect striking specificities in their function.…”

Section: Concluding Remarks and Future Perspectivesmentioning

confidence: 99%

Lesion processing: high-fidelity versus lesion-bypass DNA polymerases

Broyde¹,

Wang²,

Rechkoblit³

et al. 2008

Trends in Biochemical Sciences

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When a high-fidelity DNA polymerase encounters certain DNA-damage sites, its progress can be stalled and one or more lesion-bypass polymerases are recruited to transit the lesion. Here, we consider two representative types of lesions: (i) 7,8-dihydro-8-oxogua-nine (8-oxoG), a small, highly prevalent lesion caused by oxidative damage; and (ii) bulky lesions derived from the environmental pre-carcinogen benzo [a]pyrene, in the high-fidelity DNA polymerase Bacillus fragment (BF) from Bacillus stearothermophilus and in the lesion-bypass DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. The tight fit of the BF polymerase around the nascent base pair contrasts with the more spacious, solvent-exposed active site of Dpo4, and these differences in architecture result in distinctions in their respective functions: one-step versus stepwise polymerase translocation, mutagenic versus accurate bypass of 8-oxoG, and polymerase stalling versus mutagenic bypass at bulky benzo[a]pyrene-derived lesions. Lesions and DNA polymerasesUntil 1999, it was widely understood that bacteria have three DNA polymerases and mammals have five [1]. In 1999, however, an entirely new category of polymerases was discovered in prokaryotes and eukaryotes [2-4] -the lesion-bypass DNA polymerases. The function of lesion-bypass polymerases is to replicate past lesion-containing DNA templates in a process called translesion synthesis [5][6][7]. Now at least 16 DNA polymerases are known in eukaryotes [8] and five in bacteria [9]. Furthermore, since 1999, crystal structures of DNA polymerases, including those containing lesions, have provided new structural insights into the mechanistic aspects of translesion synthesis and polymerase stalling associated with DNA lesions. For a review of the structures and mechanisms of DNA polymerases up to the year 2005, see Ref.[10].Here, we discuss the structural and functional features of representative high-fidelity and lesion-bypass DNA polymerases (Box 1) and how these features affect the processing of certain forms of DNA damage. The lesions considered are derived from reactions of intermediates produced by endogenous sources or from the metabolic activation of environmental genotoxic

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Human DNA Polymerase κ Encircles DNA: Implications for Mismatch Extension and Lesion Bypass

Cited by 211 publications

References 47 publications

A structural gap in Dpo4 supports mutagenic bypass of a major benzo[ a ]pyrene dG adduct in DNA through template misalignment

A structural gap in Dpo4 supports mutagenic bypass of a major benzo[ a ]pyrene dG adduct in DNA through template misalignment

Variants of mouse DNA polymerase κ reveal a mechanism of efficient and accurate translesion synthesis past a benzo[ a ]pyrene dG adduct

Lesion processing: high-fidelity versus lesion-bypass DNA polymerases

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