A Structural Solution for the DNA Polymerase λ-Dependent Repair of DNA Gaps with Minimal Homology

García‐Díaz, Miguel; Bębenek, Katarzyna; Krahn, J.M.; Blanco, Luis; Kunkel, Thomas A.; Pedersen, Lars C.

doi:10.1016/s1097-2765(04)00061-9

Cited by 126 publications

(168 citation statements)

References 53 publications

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“…This interpretation is supported by in vitro analyses of Pol4 and the related mammalian Pol X NHEJ polymerases, Pol μ and Pol λ, including extensive structural data, which have revealed several unique features of the Pol X polymerases [17,24,[29][30][31][32][33][34]. For example, the Pol X NHEJ polymerases make fewer template strand contacts [24] and have a propensity toward strand slippage [17,30,31], mismatch extension [32] and lesion bypass [33], observations consistent with a reduced dependence on a stable primer-template pairing. Indeed, a "gradient" of polymerase properties has been described in which Pol μ has the remarkable ability to catalyze template-dependent synthesis without any initial primer-template pairing [34].…”

Section: Discussionmentioning

confidence: 74%

“…Thus, we infer that Pol4 achieves a productive catalytic complex by bridging the two sides of one or both DSB strands to overcome the missing contribution of base stacking to duplex stability ( Figure 6B). Because of the extent of DNA bound by the polymerase [24,29], it is highly unlikely that NHEJ structural components such as Ku or MRX can provide this level of bridging; it must come from the polymerase itself. Crystal structures of Pol λ, the mammalian polymerase most related to Pol4, provide a first insight into the interactions that likely facilitate this bridging, which, not surprisingly, include extensive base stacking.…”

Section: Discussionmentioning

confidence: 99%

“…As previously observed [9], pol4-KK247∷248RR cells showed a mild defect at rejoining a DSB containing simple Pol4-dependent gaps ( Figure 3A, first joint). Since this joint does not require lyase activity, this is likely due to destabilizing effects of the mutation on the 8 kDa domain, which binds the downstream 5' terminus in Pol X polymerases [24]. Most importantly, the pol4-KK247∷248RR strain showed only a similar minor defect at the joint with a gap and 5' dRP ( Figure 3B, first joint), unlike the severe defect seen in the polymerase mutant.…”

Section: Pol4 Lyase Activity Is Not Required At Dsbs With 5' Drp Terminimentioning

confidence: 96%

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Evidence that base stacking potential in annealed 3′ overhangs determines polymerase utilization in yeast nonhomologous end joining

Daley

Wilson

2008

DNA Repair

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Nonhomologous end joining (NHEJ) directly rejoins DNA double-strand breaks (DSBs) when recombination is not possible. In Saccharomyces cerevisiae, the DNA polymerase Pol4 is required for gap filling when a short 3' overhang must prime DNA synthesis. Here, we examined further end variations to test specific hypotheses regarding Pol4 usage in NHEJ in vivo. Surprisingly, Pol4 dependence at 3' overhangs was reduced when a nonhomologous 5' flap nucleotide was present across from the gap, even though the mismatched nucleotide was resynthesized, not incorporated. In contrast, a gap with a 5' deoxyribosephosphate (dRP) was as Pol4-dependent as a gap with a 5' phosphate, demonstrating the importance of the downstream base in relaxing the Pol4 requirement. Combined with prior observations of Pol4-independent NHEJ of nicks with 5' hydroxyls, we suggest that base stacking interactions across the broken strands can stabilize a joint, allowing another polymerase to substitute for Pol4. This model predicts that a unique function of Pol4 is to actively stabilize template strands that lack stacking continuity. We also explored whether NHEJ end processing can occur via short and long patch pathways analogous to base excision repair. Results demonstrated that 5' dRPs could be removed in the absence of Pol4 lyase activity. The 5' flap endonuclease Rad27 was not required for repair in this or any situation tested, indicating that still other NHEJ 5' nucleases must exist.

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Section: Discussionmentioning

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Section: Pol4 Lyase Activity Is Not Required At Dsbs With 5' Drp Terminimentioning

confidence: 96%

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Evidence that base stacking potential in annealed 3′ overhangs determines polymerase utilization in yeast nonhomologous end joining

Daley

Wilson

2008

DNA Repair

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“…The structure of the catalytic core of human Pol λ has been extensively characterized [9,10,[19][20][21][22]. Although structurally similar to Pol β, Pol λ displays some unique structural differences.…”

Section: Introductionmentioning

confidence: 99%

Role of the catalytic metal during polymerization by DNA polymerase lambda

et al. 2007

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The incorporation of dNMPs into DNA by polymerases involves a phosphoryl transfer reaction hypothesized to require two divalent metal ions. Here we investigate this hypothesis using as a model human DNA polymerase λ (Pol λ), an enzyme suggested to be activated in vivo by manganese. We report the crystal structures of four complexes of human Pol λ. In a 1.9Å structure of Pol λ containing a 3′ OH and the non-hydrolyzable analog dUpnpp, a non-catalytic Na + ion occupies the site for metal A and the ribose of the primer-terminal nucleotide is found in a conformation that positions the acceptor 3'OH out of line with the α-phosphate and the bridging oxygen of the pyrophosphate leaving group. Soaking this crystal in MnCl 2 yielded a 2.0Å structure with Mn 2+ occupying the site for metal A. In the presence of Mn 2+ , the conformation of the ribose is C3' endo and the 3'-oxygen is in line with the leaving oxygen, at a distance from the phosphorus atom of the α-phosphate (3.69 Å) consistent with and supporting a catalytic mechanism involving two divalent metal ions. Finally, soaking with MnCl 2 converted a pre-catalytic Pol λ/Na + complex with unreacted dCTP in the active site into a product complex via catalysis in the crystal. These data provide pre-and post-transition state information and outline in a single crystal the pathway for the phosphoryl transfer reaction carried out by DNA polymerases.

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“…Its cellular function is to fill short gaps in DNA generated during base excision repair and nonhomologous end joining of double strand breaks in DNA. We previously described several structures of Pol λ that provide information on how this polymerase incorporates a correct dNTP (17)(18)(19). In addition, we described structures of Pol λ containing single unpaired nucleotides in the template strand upstream of the active site (20,21).…”

mentioning

confidence: 99%

Replication infidelity via a mismatch with Watson–Crick geometry

Bębenek

Pedersen

Kunkel

2011

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

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In describing the DNA double helix, Watson and Crick suggested that “spontaneous mutation may be due to a base occasionally occurring in one of its less likely tautomeric forms.” Indeed, among many mispairing possibilities, either tautomerization or ionization of bases might allow a DNA polymerase to insert a mismatch with correct Watson–Crick geometry. However, despite substantial progress in understanding the structural basis of error prevention during polymerization, no DNA polymerase has yet been shown to form a natural base–base mismatch with Watson–Crick-like geometry. Here we provide such evidence, in the form of a crystal structure of a human DNA polymerase λ variant poised to misinsert dGTP opposite a template T. All atoms needed for catalysis are present at the active site and in positions that overlay with those for a correct base pair. The mismatch has Watson–Crick geometry consistent with a tautomeric or ionized base pair, with the pH dependence of misinsertion consistent with the latter. The results support the original idea that a base substitution can originate from a mismatch having Watson–Crick geometry, and they suggest a common catalytic mechanism for inserting a correct and an incorrect nucleotide. A second structure indicates that after misinsertion, the now primer-terminal G•T mismatch is also poised for catalysis but in the wobble conformation seen in other studies, indicating the dynamic nature of the pathway required to create a mismatch in fully duplex DNA.

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A Structural Solution for the DNA Polymerase λ-Dependent Repair of DNA Gaps with Minimal Homology

Cited by 126 publications

References 53 publications

Evidence that base stacking potential in annealed 3′ overhangs determines polymerase utilization in yeast nonhomologous end joining

Evidence that base stacking potential in annealed 3′ overhangs determines polymerase utilization in yeast nonhomologous end joining

Role of the catalytic metal during polymerization by DNA polymerase lambda

Replication infidelity via a mismatch with Watson–Crick geometry

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