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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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Related in: MedlinePlus

The NCS 2-fold axes in pol θ crystals are visualized and compared. (a) The ASU of the pol θ THF–ddATP model contains four protein-DNA complexes. Assembling the ASU requires two skew NCS 2-fold axes (dotted lines). (b) The NCS 2-fold relating chains C and D passes near insert 3 of the palm (pink hues) subdomain, adjacent to the N-terminal (grey shades) subdomain (c) The 5’-template DNA propagated NCS 2-fold relates chains A and B, passing near the fingers (blue hues) and thumb (green hues) subdomains. (d) Crystal packing in the dCMP–ddGTP crystal form is depicted, showing that the two molecules per ASU pack differently from the four molecules of the THF–ddATP crystal form (described in panel a). The identical NCS 2-fold axes are observed, but parallel to the 21 screw along cell edge c. The dimer at the right of the panel is generated by a crystallographic translation of the unit cell (green rectangular prism) along the b cell axis. The dimer at the bottom left is generated by the 21 screw along cell axis c (vertical axis), which in the context of the 5’-template DNA propagated NCS 2-fold axis gives rise to the NCS translation.
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Figure 8: The NCS 2-fold axes in pol θ crystals are visualized and compared. (a) The ASU of the pol θ THF–ddATP model contains four protein-DNA complexes. Assembling the ASU requires two skew NCS 2-fold axes (dotted lines). (b) The NCS 2-fold relating chains C and D passes near insert 3 of the palm (pink hues) subdomain, adjacent to the N-terminal (grey shades) subdomain (c) The 5’-template DNA propagated NCS 2-fold relates chains A and B, passing near the fingers (blue hues) and thumb (green hues) subdomains. (d) Crystal packing in the dCMP–ddGTP crystal form is depicted, showing that the two molecules per ASU pack differently from the four molecules of the THF–ddATP crystal form (described in panel a). The identical NCS 2-fold axes are observed, but parallel to the 21 screw along cell edge c. The dimer at the right of the panel is generated by a crystallographic translation of the unit cell (green rectangular prism) along the b cell axis. The dimer at the bottom left is generated by the 21 screw along cell axis c (vertical axis), which in the context of the 5’-template DNA propagated NCS 2-fold axis gives rise to the NCS translation.

Mentions: The pol θ ASU in the Ca2+ crystal form contains 4 protein-DNA complexes, assembled as a dimer of 2-fold dimers (Fig. 8a). Two dissimilar types of contacts mediated packing of the DNA ends distal from the active site: The 3’ ends of each template DNA strand provide a dGMP overhang, two of which stack against conserved tryptophan residues (W1907) of adjacent protein molecules. The remaining template 3’ ends appear in close proximity, forming inter-DNA contacts outside the molecular footprint of pol θ.


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 NCS 2-fold axes in pol θ crystals are visualized and compared. (a) The ASU of the pol θ THF–ddATP model contains four protein-DNA complexes. Assembling the ASU requires two skew NCS 2-fold axes (dotted lines). (b) The NCS 2-fold relating chains C and D passes near insert 3 of the palm (pink hues) subdomain, adjacent to the N-terminal (grey shades) subdomain (c) The 5’-template DNA propagated NCS 2-fold relates chains A and B, passing near the fingers (blue hues) and thumb (green hues) subdomains. (d) Crystal packing in the dCMP–ddGTP crystal form is depicted, showing that the two molecules per ASU pack differently from the four molecules of the THF–ddATP crystal form (described in panel a). The identical NCS 2-fold axes are observed, but parallel to the 21 screw along cell edge c. The dimer at the right of the panel is generated by a crystallographic translation of the unit cell (green rectangular prism) along the b cell axis. The dimer at the bottom left is generated by the 21 screw along cell axis c (vertical axis), which in the context of the 5’-template DNA propagated NCS 2-fold axis gives rise to the NCS translation.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: The NCS 2-fold axes in pol θ crystals are visualized and compared. (a) The ASU of the pol θ THF–ddATP model contains four protein-DNA complexes. Assembling the ASU requires two skew NCS 2-fold axes (dotted lines). (b) The NCS 2-fold relating chains C and D passes near insert 3 of the palm (pink hues) subdomain, adjacent to the N-terminal (grey shades) subdomain (c) The 5’-template DNA propagated NCS 2-fold relates chains A and B, passing near the fingers (blue hues) and thumb (green hues) subdomains. (d) Crystal packing in the dCMP–ddGTP crystal form is depicted, showing that the two molecules per ASU pack differently from the four molecules of the THF–ddATP crystal form (described in panel a). The identical NCS 2-fold axes are observed, but parallel to the 21 screw along cell edge c. The dimer at the right of the panel is generated by a crystallographic translation of the unit cell (green rectangular prism) along the b cell axis. The dimer at the bottom left is generated by the 21 screw along cell axis c (vertical axis), which in the context of the 5’-template DNA propagated NCS 2-fold axis gives rise to the NCS translation.
Mentions: The pol θ ASU in the Ca2+ crystal form contains 4 protein-DNA complexes, assembled as a dimer of 2-fold dimers (Fig. 8a). Two dissimilar types of contacts mediated packing of the DNA ends distal from the active site: The 3’ ends of each template DNA strand provide a dGMP overhang, two of which stack against conserved tryptophan residues (W1907) of adjacent protein molecules. The remaining template 3’ ends appear in close proximity, forming inter-DNA contacts outside the molecular footprint of pol θ.

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