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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) Structure-based sequence alignment illustrates the conservation of K2181, R2202, and R2254 (blue triangles) in multicellular organisms. Pol θ, mus308, and pol ν conserve an arginine or lysine at position 2254, whereas bacterial polymerases retain a hydrophobic side chain. Only pol θ-like enzymes (top nine proteins) conserve basic residues at positions 2181 and 2202. (b) Superposition of pol θ (warm pink), Taq (1QSY32, cyan), and Klenow (1KLN42, light grey) illustrates how insertion loop 2 (yellow) departs from the canonical family A polymerase fold. R2254 in human pol θ contacts the primer 3’-terminal (n-1) phosphate. (c) The palm (warm pink) and specialized thumb subdomain (green) of pol θ are illustrated to display unique contacts (black dashes) to the primer DNA (orange sticks). In addition to R2254 at the n-1 phosphate, R2202 contacts the n-2 and n-3 phosphates of the primer DNA. K2181 establishes a putative salt bridge to the n-5 phosphate, just after insertion loop 1 (brown).
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Figure 2: (a) Structure-based sequence alignment illustrates the conservation of K2181, R2202, and R2254 (blue triangles) in multicellular organisms. Pol θ, mus308, and pol ν conserve an arginine or lysine at position 2254, whereas bacterial polymerases retain a hydrophobic side chain. Only pol θ-like enzymes (top nine proteins) conserve basic residues at positions 2181 and 2202. (b) Superposition of pol θ (warm pink), Taq (1QSY32, cyan), and Klenow (1KLN42, light grey) illustrates how insertion loop 2 (yellow) departs from the canonical family A polymerase fold. R2254 in human pol θ contacts the primer 3’-terminal (n-1) phosphate. (c) The palm (warm pink) and specialized thumb subdomain (green) of pol θ are illustrated to display unique contacts (black dashes) to the primer DNA (orange sticks). In addition to R2254 at the n-1 phosphate, R2202 contacts the n-2 and n-3 phosphates of the primer DNA. K2181 establishes a putative salt bridge to the n-5 phosphate, just after insertion loop 1 (brown).

Mentions: Insert 2 (residues R2254-S2313) departs the palm subdomain near the primer terminus binding site to join the thumb subdomain, where electron density for two β-strands was observed exiting the active site. These strands constitute the most highly conserved fragments of insertion loop 2 in pol θ and mus308-like proteins, from which a highly retained basic residue (R2254 in human pol θ; Fig. 2a) contacts the 3’-terminal phosphate of the primer DNA (Fig. 2b and Supplementary Fig. 2). A second contact of interest involving R2254 appeared only in the Mg2+ crystal form, where the O-helix has closed farther towards the active site. These subtle shifts reposition the acidic C-terminal end of the O-helix, from which a well conserved aspartate residue (D2376) engages in a salt bridge with R2254 of insertion loop 2 (Fig. 1e), pinning the guanidinium moiety between the terminal phosphate and O-helix. Unlike insert 3, which resides on the opposite site of the protein from the primer–template DNA, insert 2 is poised to align a poorly matched primer terminus for nucleophilic attack. Both inserts 2 and 3 have been shown necessary for TLS by pol θ, with insert 2 required for single-stranded oligonucleotide extension10,11.


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) Structure-based sequence alignment illustrates the conservation of K2181, R2202, and R2254 (blue triangles) in multicellular organisms. Pol θ, mus308, and pol ν conserve an arginine or lysine at position 2254, whereas bacterial polymerases retain a hydrophobic side chain. Only pol θ-like enzymes (top nine proteins) conserve basic residues at positions 2181 and 2202. (b) Superposition of pol θ (warm pink), Taq (1QSY32, cyan), and Klenow (1KLN42, light grey) illustrates how insertion loop 2 (yellow) departs from the canonical family A polymerase fold. R2254 in human pol θ contacts the primer 3’-terminal (n-1) phosphate. (c) The palm (warm pink) and specialized thumb subdomain (green) of pol θ are illustrated to display unique contacts (black dashes) to the primer DNA (orange sticks). In addition to R2254 at the n-1 phosphate, R2202 contacts the n-2 and n-3 phosphates of the primer DNA. K2181 establishes a putative salt bridge to the n-5 phosphate, just after insertion loop 1 (brown).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: (a) Structure-based sequence alignment illustrates the conservation of K2181, R2202, and R2254 (blue triangles) in multicellular organisms. Pol θ, mus308, and pol ν conserve an arginine or lysine at position 2254, whereas bacterial polymerases retain a hydrophobic side chain. Only pol θ-like enzymes (top nine proteins) conserve basic residues at positions 2181 and 2202. (b) Superposition of pol θ (warm pink), Taq (1QSY32, cyan), and Klenow (1KLN42, light grey) illustrates how insertion loop 2 (yellow) departs from the canonical family A polymerase fold. R2254 in human pol θ contacts the primer 3’-terminal (n-1) phosphate. (c) The palm (warm pink) and specialized thumb subdomain (green) of pol θ are illustrated to display unique contacts (black dashes) to the primer DNA (orange sticks). In addition to R2254 at the n-1 phosphate, R2202 contacts the n-2 and n-3 phosphates of the primer DNA. K2181 establishes a putative salt bridge to the n-5 phosphate, just after insertion loop 1 (brown).
Mentions: Insert 2 (residues R2254-S2313) departs the palm subdomain near the primer terminus binding site to join the thumb subdomain, where electron density for two β-strands was observed exiting the active site. These strands constitute the most highly conserved fragments of insertion loop 2 in pol θ and mus308-like proteins, from which a highly retained basic residue (R2254 in human pol θ; Fig. 2a) contacts the 3’-terminal phosphate of the primer DNA (Fig. 2b and Supplementary Fig. 2). A second contact of interest involving R2254 appeared only in the Mg2+ crystal form, where the O-helix has closed farther towards the active site. These subtle shifts reposition the acidic C-terminal end of the O-helix, from which a well conserved aspartate residue (D2376) engages in a salt bridge with R2254 of insertion loop 2 (Fig. 1e), pinning the guanidinium moiety between the terminal phosphate and O-helix. Unlike insert 3, which resides on the opposite site of the protein from the primer–template DNA, insert 2 is poised to align a poorly matched primer terminus for nucleophilic attack. Both inserts 2 and 3 have been shown necessary for TLS by pol θ, with insert 2 required for single-stranded oligonucleotide extension10,11.

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