DNA polymerase μ (Pol μ) is the only template-dependent human DNA polymerase capable of repairing double-strand DNA breaks (DSBs) with unpaired 3′ ends in nonhomologous end joining (NHEJ). To probe this function, we structurally characterized Pol μ's catalytic cycle for single-nucleotide incorporation. These structures indicate that, unlike other template-dependent DNA polymerases, Pol μ shows no large-scale conformational changes in protein subdomains, amino acid side chains or DNA upon dNTP binding or catalysis. Instead, the only major conformational change is seen earlier in the catalytic cycle, when the flexible loop 1 region repositions upon DNA binding. Pol μ variants with changes in loop 1 have altered catalytic properties and are partially defective in NHEJ. The results indicate that specific loop 1 residues contribute to Pol μ's unique ability to catalyze template-dependent NHEJ of DSBs with unpaired 3′ ends.
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We thank G. Mueller and B. Beard for critical reading of the manuscript. This research was supported by the Division of Intramural Research of the US National Institute of Environmental Health Sciences, National Institutes of Health (NIH) grants ES102645 (L.C.P.), and ES065070 (T.A.K.) and by NIH grant CA097096 (D.A.R.). Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences contract W-31-109-Eng-38.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Comparison of wild-type full-length and catalytic-domain (Pro138–Ala494) constructs of hPol μ in single-nucleotide gap-filling.
A steady-state polymerase activity assay was performed in order to compare wildtype full-length or truncated Pol μ constructs (5nM), on a single-nucleotide gapped DNA substrate (no enzyme control, C). The percentage of primer extension was calculated for each construct, at two different time points. The two constructs behaved indistinguishably in this assay.
(a) Comparison of the X-ray crystal structures of mouse Pol μ (PDB ID code 2IHM1, purple) and mouse TdT (PDB ID code 1JMS2, orange). The relative positions of Loop 1 (green, from mTdT) and Loop 2 (red, from mPol μ) are displayed. (b) Ribbon diagram showing the ordered and disordered regions of Loop 2 in mTdT (orange) and mPol μ (red). (c) ClustalW3 sequence alignments of Loop1 and Loop 2 in mammalian orthologs of Pol μ. Loop1 is boxed in green. Regions of decreased sequence conservation in Loop2 are boxed in red, and were subsequently deleted by site-directed mutagenesis. His363 (magenta), Met382 (green), and Phe385 (cyan) are clearly marked. (d) ClustalW sequence alignments of Loop 2 in mammalian orthologs of TdT. Loop1 is boxed in green, and regions that are structurally homologous to the deleted region of Loop 2 in hPol μ are boxed in red. Phe401 is marked in cyan. (e) Structure of mPol μ (purple), displaying the location of Loop 2 (red), distal from the active site (marked by the incoming nucleotide, cyan), and the DNA binding cleft (khaki ribbon and surface rendering). (f) Structure of engineered Loop 2 from hPol μ Δ2 binary complex (blue), compared to mPol μ (purple). Residues Pro398-Pro410 were deleted, and β-strands 4 and 5 fused by addition of a glycine residue (labeled Gly410 and marked by an asterisk). All structural figures were generated using PyMOL4.
(a) Comparison of wildtype and Δ2 variant hPol μ template-dependent synthesis activity on a single nucleotide gapped DNA substrate. (b) Comparison of wildtype and Δ2 hPol μ template-independent synthesis activity on a single-stranded oligo-dT DNA substrate. (c) Comparison of wildtype and hPol μ Δ2 during in vitro NHEJ assays. Top, schematic diagram illustrating the structure of the DNA substrates used in the NHEJ assay. Bottom, ligation products of NHEJ synthesized by either wildtype or hPol μ Δ2.
Supplementary Figure 4 Implications of loop 1 flexibility for substrate stabilization during DSB repair by NHEJ.
(a) Model of a noncomplementary DSB substrate (purple) bound to the protein component of the hPol μ Δ2 pre-catalytic ternary complex (orange). The primer (strand P) terminus (blue) is unpaired, opposite the discontinuity in the template strand (strand T). The incoming nucleotide (cyan) is correctly paired opposite the templating base (magenta). Location of the disordered Loop1 is marked in green. (b) Hypothetical Loop1 conformations were manually generated for the single-nucleotide gapped ternary complex (green) and for a noncomplementary DSB substrate (red).
(a) Uncropped gel from Figure 5b. (b) Uncropped gel from Figure 5c (top panel). (c) Uncropped gel from Figure 5c (bottom panel). All wells in (b) and (c) also contain human Ku (10 nM) and XRCC4/Ligase IV complex (20 nM) in addition to the indicated Family X polymerase (0.5 nM).
The vestigial thrombin protease cleavage site from the parental pGEX-4T3 expression vector is highlighted in blue. The Tobacco Etch Virus (TEV) protease cleavage site is shown in green, with a green line clearly delineating the actual site of cleavage. The BamHI restriction site between the two regions encoding the thrombin and TEV protease sites has been deactivated by site directed mutagenesis (blue asterisk), a silent mutation which does not affect the protein sequence. The multicloning site from the pMALX expression vector is shown5, with the pertinent restriction sites marked. Three TGA stop codons (red) are situated immediately downstream of the multicloning site, one in each reading frame. Locations of sequencing primers 5'pGEX and 3'pGEX are shown with directional arrows.
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Moon, A., Pryor, J., Ramsden, D. et al. Sustained active site rigidity during synthesis by human DNA polymerase μ. Nat Struct Mol Biol 21, 253–260 (2014). https://doi.org/10.1038/nsmb.2766
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