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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: 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.

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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(a) Primer extension assays utilizing a single-stranded substrate show that pol θ variants K2181A, R2202A, R2254A and R2254V are all able to catalyze extension of a single-stranded oligonucleotide after 10 min in the indicated sequence context. (b) A primer extension assay in an alternate sequence context shows that variants of R2202 and R2254 produce shorter products when forced to utilize pyrimidines. In this sequence context, K2202A, R2254A, and R2254V appear unable to utilize dTTP and show reduced incorporation of dCTP compared to the parental enzyme.
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Figure 7: (a) Primer extension assays utilizing a single-stranded substrate show that pol θ variants K2181A, R2202A, R2254A and R2254V are all able to catalyze extension of a single-stranded oligonucleotide after 10 min in the indicated sequence context. (b) A primer extension assay in an alternate sequence context shows that variants of R2202 and R2254 produce shorter products when forced to utilize pyrimidines. In this sequence context, K2202A, R2254A, and R2254V appear unable to utilize dTTP and show reduced incorporation of dCTP compared to the parental enzyme.

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)

(a) Primer extension assays utilizing a single-stranded substrate show that pol θ variants K2181A, R2202A, R2254A and R2254V are all able to catalyze extension of a single-stranded oligonucleotide after 10 min in the indicated sequence context. (b) A primer extension assay in an alternate sequence context shows that variants of R2202 and R2254 produce shorter products when forced to utilize pyrimidines. In this sequence context, K2202A, R2254A, and R2254V appear unable to utilize dTTP and show reduced incorporation of dCTP compared to the parental enzyme.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: (a) Primer extension assays utilizing a single-stranded substrate show that pol θ variants K2181A, R2202A, R2254A and R2254V are all able to catalyze extension of a single-stranded oligonucleotide after 10 min in the indicated sequence context. (b) A primer extension assay in an alternate sequence context shows that variants of R2202 and R2254 produce shorter products when forced to utilize pyrimidines. In this sequence context, K2202A, R2254A, and R2254V appear unable to utilize dTTP and show reduced incorporation of dCTP compared to the parental enzyme.
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: 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.

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