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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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The primer extension assay demonstrates that pol θ R2254V cannot perform the bypass step of TLS, even when all nucleotides are provided (500 µM each). Pol θ and the human chaos1 allele homolog (pol θ S1977P) bypass the AP site to a full-length product, with terminal transferase activity extending the primer by one additional nucleotide. Klenow fragment exo- is able to bypass the AP site under these reaction conditions (125 nM enzyme + 250 nM primer–template), without terminal transferase activity.
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Figure 4: The primer extension assay demonstrates that pol θ R2254V cannot perform the bypass step of TLS, even when all nucleotides are provided (500 µM each). Pol θ and the human chaos1 allele homolog (pol θ S1977P) bypass the AP site to a full-length product, with terminal transferase activity extending the primer by one additional nucleotide. Klenow fragment exo- is able to bypass the AP site under these reaction conditions (125 nM enzyme + 250 nM primer–template), without terminal transferase activity.

Mentions: The human pol θ model illustrates that S1977 caps the C-terminal end of a hydrophobic helix in the vestigial exonuclease-like subdomain. In the current model, S1997 could provide a hydrogen bond to the backbone carbonyl of D1897 of loop exo1 (Fig. 3b). We generated the S1977P mutant to characterize the biochemical characteristics of the homologous human chaos1 variant. Assays aimed to evaluate TLS (Fig. 4) and single-stranded primer extension (data not shown) failed to reveal a dramatic biochemical phenotype, however. These findings support the suggestion that cellular levels of the pol θ protein are depleted in the chaos1 mice due to poor in vivo expression or stability18.


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)

The primer extension assay demonstrates that pol θ R2254V cannot perform the bypass step of TLS, even when all nucleotides are provided (500 µM each). Pol θ and the human chaos1 allele homolog (pol θ S1977P) bypass the AP site to a full-length product, with terminal transferase activity extending the primer by one additional nucleotide. Klenow fragment exo- is able to bypass the AP site under these reaction conditions (125 nM enzyme + 250 nM primer–template), without terminal transferase activity.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: The primer extension assay demonstrates that pol θ R2254V cannot perform the bypass step of TLS, even when all nucleotides are provided (500 µM each). Pol θ and the human chaos1 allele homolog (pol θ S1977P) bypass the AP site to a full-length product, with terminal transferase activity extending the primer by one additional nucleotide. Klenow fragment exo- is able to bypass the AP site under these reaction conditions (125 nM enzyme + 250 nM primer–template), without terminal transferase activity.
Mentions: The human pol θ model illustrates that S1977 caps the C-terminal end of a hydrophobic helix in the vestigial exonuclease-like subdomain. In the current model, S1997 could provide a hydrogen bond to the backbone carbonyl of D1897 of loop exo1 (Fig. 3b). We generated the S1977P mutant to characterize the biochemical characteristics of the homologous human chaos1 variant. Assays aimed to evaluate TLS (Fig. 4) and single-stranded primer extension (data not shown) failed to reveal a dramatic biochemical phenotype, however. These findings support the suggestion that cellular levels of the pol θ protein are depleted in the chaos1 mice due to poor in vivo expression or stability18.

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