DNA Polymerase λ Protects Mouse Fibroblasts against Oxidative DNA Damage and Is Recruited to Sites of DNA Damage/Repair

Braithwaite, Elena K.; Kedar, Padmini S.; Lan, Li; Polosina, Yaroslava Y.; Asagoshi, Kenjiro; Poltoratsky, Vladimir; Horton, Julie K.; Miller, Holly; Teebor, George W.; Yasui, Akira; Wilson, Samuel H.

doi:10.1074/jbc.c500256200

Cited by 107 publications

(118 citation statements)

References 32 publications

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“…Each has been shown capable of removing the 5'dRP lesion subsequent to APE1 strand cleavage [77,78] [79], confirming that pol ß is the predominant activity in mouse embryonic fibroblasts (MEFs) [79]. However, it was demonstrated that pol λ deficient MEFs are sensitive to hydrogen peroxide [80], suggesting that the participation of either pol λ or pol ι in BER may be lesion specific. Complementation studies will be required to determine the precise role, if any, of these alternate BER polymerases in cell survival following genotoxic stress.…”

Section: Gap Tailoringmentioning

confidence: 92%

A unified view of base excision repair: Lesion-dependent protein complexes regulated by post-translational modification

Almeida¹,

Sobol²

2007

DNA Repair

387

374

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Base excision repair (BER) proteins act upon a significantly broad spectrum of DNA lesions that result from endogenous and exogenous sources. Multiple sub-pathways of BER (short-path or longpatch) and newly designated DNA repair pathways (e.g., SSBR and NIR) that utilize BER proteins complicate any comprehensive understanding of BER and its role in genome maintenance, chemotherapeutic response, neurodegeneration, cancer or aging. Herein, we propose a unified model of BER, comprised of three functional processes: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation, each represented by one or more multiprotein complexes and coordinated via the XRCC1/DNA Ligase III and PARP1 scaffold proteins. BER therefore may be represented by a series of repair complexes that assemble at the site of the DNA lesion and mediates repair in a coordinated fashion involving protein-protein interactions that dictate subsequent steps or sub-pathway choice. Complex formation is influenced by post-translational protein modifications that arise from the cellular state or the DNA damage response, providing an increase in specificity and efficiency to the BER pathway. In this review, we have summarized the reported BER proteinprotein interactions and protein post-translational modifications and discuss the impact on DNA repair capacity and complex formation.

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Section: Gap Tailoringmentioning

confidence: 92%

A unified view of base excision repair: Lesion-dependent protein complexes regulated by post-translational modification

Almeida¹,

Sobol²

2007

DNA Repair

387

374

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“…Cell extracts, as indicated in figure legends, were separated by Nu-PAGE 4-12% Bis-Tris mini gel (Invitrogen), and the proteins were transferred electrophoretically to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.5% (v/v) Tween-20 (TBST) for 1 h, washed once with TBST, and incubated for 2 h with appropriately diluted primary antibody against rat pol β (18S) [18], human pol ι [20], human pol λ [21], and anti-glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (Alpha Diagnostic Intl. Inc., San Antonio, TX).…”

Section: Immunoblottingmentioning

confidence: 99%

Down-regulation of DNA polymerase β accompanies somatic hypermutation in human BL2 cell lines

et al. 2007

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Somatic hypermutation (SHM) is a fundamental process in immunoglobulin gene maturation that results in increased affinity of antibodies toward antigens. In one hypothesis explaining SHM in human B cells, the process is initiated by enzymatic deamination of cytosine to uracil in the immunoglobulin gene V-region and this in turn triggers mutation-prone forms of uracil-DNA base excision repair (BER). Yet, an uncertainty with this model is that BER of uracil-DNA in mammalian cells is generally error-free, wherein DNA polymerase β (pol β) conducts gap-filling synthesis by insertion of bases according to Watson-Crick rules. To evaluate this inconsistency, we examined pol β expression in various SHM proficient human BL2 cell line subclones. We report that expression of pol β in SHM proficient cell lines was strongly down-regulated. In contrast, in other BL2 subclones, we found that SHM was deficient and that pol β expression was much higher than in the SHM proficient subclones. We also found that overexpression of recombinant human pol β in a SHM proficient subclone abrogated its capacity for SHM. These results suggest that down-regulation of the normal BER gap-filling DNA polymerase, pol β, accompanies induced SHM in BL2 cells. This is consistent with the hypothesis that normal error-free BER must be silenced to make way for an error-prone BER process that may be required during somatic hypermutation.

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“…This flexibility was first suggested by the observation that Pol λ generates deletion errors at an extremely high rate [11], and is now thought to be a feature of the enzyme that facilitates its role in vivo. The phenotypes of mice deficient in this polymerase indicate that Pol λ plays a role in the V(D)J process of antigen gene diversification [12], and several reports implicate Pol λ in Base Excision Repair [13][14][15] and the repair of double-strand breaks through the Non-Homologous DNA End-Joining pathway [16][17][18]. It is thus interesting to examine the details of the polymerization reaction as catalyzed by Pol λ in order to understand whether specific catalytic features can account for these special properties.…”

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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DNA Polymerase λ Protects Mouse Fibroblasts against Oxidative DNA Damage and Is Recruited to Sites of DNA Damage/Repair

Cited by 107 publications

References 32 publications

A unified view of base excision repair: Lesion-dependent protein complexes regulated by post-translational modification

A unified view of base excision repair: Lesion-dependent protein complexes regulated by post-translational modification

Down-regulation of DNA polymerase β accompanies somatic hypermutation in human BL2 cell lines

Role of the catalytic metal during polymerization by DNA polymerase lambda

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