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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

(a) The overall THF–ddATP pol θ structure is shown in comparison to Taq polymerase (circle insert; PDBID 1QSY32), identifying the 5 insertion loops. Loops exo1 and exo2 (cyan) extend from the β-sheet of the exonuclease-like subdomain. Insert 1 (brown) occupies the tip of the thumb subdomain. Insert 2 (yellow) joins the thumb subdomain, adjacent to the active site. Insert 3 (purple) of the palm subdomain lies in close proximately to the exonuclease-like subdomain. The dotted lines represent regions of the loops that were not seen in the electron density map and thus not built in the crystallographic model (b) A putty representation of pol θ, in the same orientation as panel a, displays the tube radius of the backbone trace proportionally to the refined atomic displacement parameters. Peaks from an NCS averaged anomalous difference electron density map (yellow mesh, contoured at 4 σ) pinpoint the location of methionines. (c) The full-length human pol θ domain architecture schematic describes the crystallization constructs, which encompass the entire C-terminal polymerase domain (residues 1819 to 2590) and vestigial exonuclease-like domain (residues 1819 to 2090). (d) A close-up view of the pol θ active site shows ddATP opposite THF in the closed conformation. Contacts (black dashes) are mediated from the O-helix residue Q2384 to the incoming nucleobase. (e) The THF–ddATP (dark pink, dark blue, and yellow-green) and dCMP–ddGTP (lighter hues) models appear superimposed, based on palm subdomain residues. Subtle rearrangements with cognate dCMP–ddGTP in the active site reposition the C-terminal end of the O-helix, forming a putative salt bridge from R2254 of insert 2 to D2376 of the fingers. All molecular illustrations were made with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC).
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Figure 1: (a) The overall THF–ddATP pol θ structure is shown in comparison to Taq polymerase (circle insert; PDBID 1QSY32), identifying the 5 insertion loops. Loops exo1 and exo2 (cyan) extend from the β-sheet of the exonuclease-like subdomain. Insert 1 (brown) occupies the tip of the thumb subdomain. Insert 2 (yellow) joins the thumb subdomain, adjacent to the active site. Insert 3 (purple) of the palm subdomain lies in close proximately to the exonuclease-like subdomain. The dotted lines represent regions of the loops that were not seen in the electron density map and thus not built in the crystallographic model (b) A putty representation of pol θ, in the same orientation as panel a, displays the tube radius of the backbone trace proportionally to the refined atomic displacement parameters. Peaks from an NCS averaged anomalous difference electron density map (yellow mesh, contoured at 4 σ) pinpoint the location of methionines. (c) The full-length human pol θ domain architecture schematic describes the crystallization constructs, which encompass the entire C-terminal polymerase domain (residues 1819 to 2590) and vestigial exonuclease-like domain (residues 1819 to 2090). (d) A close-up view of the pol θ active site shows ddATP opposite THF in the closed conformation. Contacts (black dashes) are mediated from the O-helix residue Q2384 to the incoming nucleobase. (e) The THF–ddATP (dark pink, dark blue, and yellow-green) and dCMP–ddGTP (lighter hues) models appear superimposed, based on palm subdomain residues. Subtle rearrangements with cognate dCMP–ddGTP in the active site reposition the C-terminal end of the O-helix, forming a putative salt bridge from R2254 of insert 2 to D2376 of the fingers. All molecular illustrations were made with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC).

Mentions: To identify structural components providing for the unique enzymatic activities of pol θ, we determined the crystal structures of two DNA polymerase domain constructs. Molecular replacement31, using a ternary complex of Taq DNA polymerase (1QSY)32 as the search model, sufficed to place the four similar molecules in the crystal asymmetric unit (ASU) of the THF–ddATP complex, which captured human pol θ inserting ddATP opposite tetrahydrofuran (THF), a stable abasic (AP) site analog. Refinement33 at 3.9 Å to an RFree of 30.2 % (RWork= 24.1 %; Table 1) revealed an overall fold reminiscent of bacterial homologs—with exonuclease, thumb and fingers subdomains oriented about a right hand palm subdomain (Supplementary Table 1)—but disrupted by five unique insertion loops (Fig. 1a–c and Supplementary Table 2). The THF–ddATP complex showed clearly the nascent base pair in the polymerase active site: The strictly conserved catalytic aspartate and glutamate residues (D2330, D2540, and E2541) of the palm subdomain coordinate a divalent Ca2+ ion, associated with the triphosphate tail of the ddATP nucleotide (Fig. 1d and Supplementary Fig. 1). As a known inhibitor of DNA polymerases, Ca2+ was essential for trapping the closed complex, because the primer strand retains a 3’-hydroxyl moiety for nucleophilic attack (Supplementary Fig. 2). The highly conserved lysine (K2383) and arginine (R2379) residues of the fingers subdomain O-helix contact non-bridging oxygens of the α and γ phosphates, respectively, as seen in other closed ternary complexes of family A polymerases (Supplementary Fig. 2)34. The conserved O-helix residue Y2391 was fully displaced from its template-occluding position in open or “ajar” structures35 (Fig. 1d and Supplementary Fig. 1), demonstrating that the pol θ structure represents the first fully closed model of any family A DNA polymerase inserting adenine opposite a non-templating DNA lesion. Previously, trapping ternary complexes destabilized by templating THF has required the purine analog 5-nitro-1-indolyl-2’-deoxyribose-5’-triphosphate (5-NITP), because of its enhanced capacity for base stacking36,37. The 5-NITP and ddATP double rings show substantial overlap when the current pol θ model is superimposed onto these previous structures38.


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) The overall THF–ddATP pol θ structure is shown in comparison to Taq polymerase (circle insert; PDBID 1QSY32), identifying the 5 insertion loops. Loops exo1 and exo2 (cyan) extend from the β-sheet of the exonuclease-like subdomain. Insert 1 (brown) occupies the tip of the thumb subdomain. Insert 2 (yellow) joins the thumb subdomain, adjacent to the active site. Insert 3 (purple) of the palm subdomain lies in close proximately to the exonuclease-like subdomain. The dotted lines represent regions of the loops that were not seen in the electron density map and thus not built in the crystallographic model (b) A putty representation of pol θ, in the same orientation as panel a, displays the tube radius of the backbone trace proportionally to the refined atomic displacement parameters. Peaks from an NCS averaged anomalous difference electron density map (yellow mesh, contoured at 4 σ) pinpoint the location of methionines. (c) The full-length human pol θ domain architecture schematic describes the crystallization constructs, which encompass the entire C-terminal polymerase domain (residues 1819 to 2590) and vestigial exonuclease-like domain (residues 1819 to 2090). (d) A close-up view of the pol θ active site shows ddATP opposite THF in the closed conformation. Contacts (black dashes) are mediated from the O-helix residue Q2384 to the incoming nucleobase. (e) The THF–ddATP (dark pink, dark blue, and yellow-green) and dCMP–ddGTP (lighter hues) models appear superimposed, based on palm subdomain residues. Subtle rearrangements with cognate dCMP–ddGTP in the active site reposition the C-terminal end of the O-helix, forming a putative salt bridge from R2254 of insert 2 to D2376 of the fingers. All molecular illustrations were made with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: (a) The overall THF–ddATP pol θ structure is shown in comparison to Taq polymerase (circle insert; PDBID 1QSY32), identifying the 5 insertion loops. Loops exo1 and exo2 (cyan) extend from the β-sheet of the exonuclease-like subdomain. Insert 1 (brown) occupies the tip of the thumb subdomain. Insert 2 (yellow) joins the thumb subdomain, adjacent to the active site. Insert 3 (purple) of the palm subdomain lies in close proximately to the exonuclease-like subdomain. The dotted lines represent regions of the loops that were not seen in the electron density map and thus not built in the crystallographic model (b) A putty representation of pol θ, in the same orientation as panel a, displays the tube radius of the backbone trace proportionally to the refined atomic displacement parameters. Peaks from an NCS averaged anomalous difference electron density map (yellow mesh, contoured at 4 σ) pinpoint the location of methionines. (c) The full-length human pol θ domain architecture schematic describes the crystallization constructs, which encompass the entire C-terminal polymerase domain (residues 1819 to 2590) and vestigial exonuclease-like domain (residues 1819 to 2090). (d) A close-up view of the pol θ active site shows ddATP opposite THF in the closed conformation. Contacts (black dashes) are mediated from the O-helix residue Q2384 to the incoming nucleobase. (e) The THF–ddATP (dark pink, dark blue, and yellow-green) and dCMP–ddGTP (lighter hues) models appear superimposed, based on palm subdomain residues. Subtle rearrangements with cognate dCMP–ddGTP in the active site reposition the C-terminal end of the O-helix, forming a putative salt bridge from R2254 of insert 2 to D2376 of the fingers. All molecular illustrations were made with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC).
Mentions: To identify structural components providing for the unique enzymatic activities of pol θ, we determined the crystal structures of two DNA polymerase domain constructs. Molecular replacement31, using a ternary complex of Taq DNA polymerase (1QSY)32 as the search model, sufficed to place the four similar molecules in the crystal asymmetric unit (ASU) of the THF–ddATP complex, which captured human pol θ inserting ddATP opposite tetrahydrofuran (THF), a stable abasic (AP) site analog. Refinement33 at 3.9 Å to an RFree of 30.2 % (RWork= 24.1 %; Table 1) revealed an overall fold reminiscent of bacterial homologs—with exonuclease, thumb and fingers subdomains oriented about a right hand palm subdomain (Supplementary Table 1)—but disrupted by five unique insertion loops (Fig. 1a–c and Supplementary Table 2). The THF–ddATP complex showed clearly the nascent base pair in the polymerase active site: The strictly conserved catalytic aspartate and glutamate residues (D2330, D2540, and E2541) of the palm subdomain coordinate a divalent Ca2+ ion, associated with the triphosphate tail of the ddATP nucleotide (Fig. 1d and Supplementary Fig. 1). As a known inhibitor of DNA polymerases, Ca2+ was essential for trapping the closed complex, because the primer strand retains a 3’-hydroxyl moiety for nucleophilic attack (Supplementary Fig. 2). The highly conserved lysine (K2383) and arginine (R2379) residues of the fingers subdomain O-helix contact non-bridging oxygens of the α and γ phosphates, respectively, as seen in other closed ternary complexes of family A polymerases (Supplementary Fig. 2)34. The conserved O-helix residue Y2391 was fully displaced from its template-occluding position in open or “ajar” structures35 (Fig. 1d and Supplementary Fig. 1), demonstrating that the pol θ structure represents the first fully closed model of any family A DNA polymerase inserting adenine opposite a non-templating DNA lesion. Previously, trapping ternary complexes destabilized by templating THF has required the purine analog 5-nitro-1-indolyl-2’-deoxyribose-5’-triphosphate (5-NITP), because of its enhanced capacity for base stacking36,37. The 5-NITP and ddATP double rings show substantial overlap when the current pol θ model is superimposed onto these previous structures38.

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