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Human DNA polymerase θ grasps the primer terminus to mediate DNA repair.

Zahn KE, Averill AM, Aller P, Wood RD, Doublié S - Nat. Struct. Mol. Biol. (2015)

Bottom Line: Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization.The second structure describes a cognate ddGTP complex.These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, USA.

ABSTRACT
DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break-inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction with the 3'-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.

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Related in: MedlinePlus

Bands from the primer extension assays shown in Fig. 5 were quantified in order to plot the average extension by pol θ thumb subdomain variants opposite an AP site, Tg, and an undamaged template.
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Figure 6: Bands from the primer extension assays shown in Fig. 5 were quantified in order to plot the average extension by pol θ thumb subdomain variants opposite an AP site, Tg, and an undamaged template.

Mentions: We designed the R2254V variant to evaluate how the conserved basic residue of insertion loop 2 contributes to pol θ’s activity on single-stranded DNA oligonucleotides, and bypass of AP sites or Tg lesions. Family A DNA polymerases from bacteria conserve a hydrophobic amino acid (valine or isoleucine) at the equivalent position of R2254 (Fig. 2a), and the R2254V variant therefore mimics these bacterial enzymes. Pol θ R2254V, although active on double-stranded DNA, failed to bypass AP sites or Tg (Figs. 4–6), and was marginally hindered during extension of unannealed single-stranded DNA oligonucleotides, especially when provided with only pyrimidine nucleotides (Fig. 7a,b). The salt bridge from R2254 to the primer 3’-terminal phosphate appears essential in compensating for interactions missing from the templating strand, due to DNA lesions or distorted base pairing.


Human DNA polymerase θ grasps the primer terminus to mediate DNA repair.

Zahn KE, Averill AM, Aller P, Wood RD, Doublié S - Nat. Struct. Mol. Biol. (2015)

Bands from the primer extension assays shown in Fig. 5 were quantified in order to plot the average extension by pol θ thumb subdomain variants opposite an AP site, Tg, and an undamaged template.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4385486&req=5

Figure 6: Bands from the primer extension assays shown in Fig. 5 were quantified in order to plot the average extension by pol θ thumb subdomain variants opposite an AP site, Tg, and an undamaged template.
Mentions: We designed the R2254V variant to evaluate how the conserved basic residue of insertion loop 2 contributes to pol θ’s activity on single-stranded DNA oligonucleotides, and bypass of AP sites or Tg lesions. Family A DNA polymerases from bacteria conserve a hydrophobic amino acid (valine or isoleucine) at the equivalent position of R2254 (Fig. 2a), and the R2254V variant therefore mimics these bacterial enzymes. Pol θ R2254V, although active on double-stranded DNA, failed to bypass AP sites or Tg (Figs. 4–6), and was marginally hindered during extension of unannealed single-stranded DNA oligonucleotides, especially when provided with only pyrimidine nucleotides (Fig. 7a,b). The salt bridge from R2254 to the primer 3’-terminal phosphate appears essential in compensating for interactions missing from the templating strand, due to DNA lesions or distorted base pairing.

Bottom Line: Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization.The second structure describes a cognate ddGTP complex.These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, USA.

ABSTRACT
DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break-inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction with the 3'-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.

Show MeSH
Related in: MedlinePlus