The double-strand break (DSB) repair pathway called microhomology-mediated end-joining (MMEJ) is thought to be dependent on DNA polymerase theta (Polθ) and occur independently of nonhomologous end-joining (NHEJ) factors. An unresolved question is whether MMEJ is facilitated by a single Polθ-mediated end-joining pathway or consists of additional undiscovered pathways. We find that human X-family Polλ, which functions in NHEJ, additionally exhibits robust MMEJ activity like Polθ. Polλ promotes MMEJ in mammalian cells independently of essential NHEJ factors LIG4/XRCC4 and Polθ, which reveals a distinct Polλ-dependent MMEJ mechanism. X-ray crystallography employing in situ photo-induced DSB formation captured Polλ in the act of stabilizing a microhomology-mediated DNA synapse with incoming nucleotide at 2.0 Å resolution and reveals how Polλ performs replication across a DNA synapse joined by minimal base-pairing. Last, we find that Polλ is semisynthetic lethal with BRCA1 and BRCA2. Together, these studies indicate Polλ MMEJ as a distinct DSB repair mechanism.
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This report is dedicated to Samuel H. Wilson who passed away during the course of these studies. We thank B. Copeland (National Institute of Environmental Health Sciences) for providing Polκ and M. O’Donnell (Rockefeller University) for providing recombinant yeast Polε. We are grateful to Dr. Wilson (NIEH) for providing recombinant human Polλ, Polμ, and Polβ. This research was supported by National Institute of Health grants 1R01GM130889 and 1R01GM137124 to R.T.P., and DOD W81XWH-18-1-0148 and Career Enhancement Program grant from the Yale Head and Neck Cancer SPORE to S.A.
R.T.P. is a cofounder and chief scientific officer of Recombination Therapeutics, LLC. The other authors declare no competing interests.
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Extended Data Fig. 1 Controls for Pol activity on primer-templates and substrates containing 3’ ssDNA.
a, Denaturing gels showing extension of the indicated primer-template by the indicated Pols using identical conditions. Polμ is known to require a downstream ssDNA strand for optimal activity primer extension activity along a short gap. 20 nM Pol concentrations were used. b, Schematic of DNA templates. Microhomology indicated as red text. c, Non-denaturing gel showing Polλ MMEJ as a positive control for its activity (left panel). Denaturing gels showing extension of the indicated templates in the presence of all 4 dNTPs by the indicated Pols (middle and right panel). 20 nM Pol concentrations were used. d, Schematic showing the respective activities of Polθ and Polλ on the indicated templates. although both enzymes perform MMEJ (top), only Polθ exhibits ssDNA extension due to its snap-back replication activity.
a, Denaturing gel showing extension of a pssDNA substrate containing 2 bp of microhomology in the presence of dCTP. Polλ but not Polμ performs addition of dCMP on the indicated substrate. Microhomology indicated as red text. 20 nM Pol concentrations were used. b, Non-denaturing gel showing MMEJ activity by the indicated Pols on the indicated pssDNA containing 6 bp of microhomology (red text). Polλ performs MMEJ whereas Polβ does not. Reactions were performed in duplicate. 20 nM Pol concentrations were used.
Extended Data Fig. 3 Supplemental data for protein expression, genetic engineering, and MMEJ activity.
a. RT qPCR analysis of Polθ expression. mRNA levels were corrected with internal control for Actin in siRNA-treated cells used in Fig. 3b, d as well as normalized to non-targeting siRNA (siControl = 1). Data represent mean. n = 1 experiment with triplicate for each condition ±SEM. b. gRNA sequence used to generate POLL−/− HEK293T cells via CRISPR-Cas9 engineering. Schematic representation of three isoforms of human Polλ with protein domains as well as location of gRNA sequence (red) is indicated. The genome sequence flanking the gRNA sequence (red) is shown in gray. POLL −/− clone # T2 was generated by CRISPR-Cas9 engineering and carries 7 bp deletion in both alleles. Sequence of the region harboring the 7 bp deletion is indicated in blue. c. Bar plot showing relative GFP following overexpression of indicated plasmids and co- transfection of left and right MMEJ reporter DNA constructs in HEK293T cells. GFP+ frequencies are normalized to transfection efficiency. Data represent mean. n = 1 experiment with triplicates for each condition, +/- s.e.m. Bottom panel: Immunoblot showing abundance of protein. d. gRNA sequence used to generate LIG4 −/− HEK293T cells (top) and XRCC4 -/- HEK293T cells (bottom) via CRISPR-Cas9 engineering. Schematic representation of human Lig4 (top) and Xrcc4 (bottom) with protein domains as well as location of gRNA sequence is indicated (red). e. Same as in Fig. 3f in XRCC4−/− HCT116 cells. Data represent mean. n = 1 experiment with triplicate for each condition, +/− s.e.m. Bottom panel: Immunoblot showing abundance of protein. f. Western blot of Polλ (top) and Gapdh (bottom) following transfection of either Polλ siRNA or siControl in DLD1 BRCA2+/+ (left) and DLD1 BRCA2 −/− cells (right).
a. Plot showing percentage of colonies relative to control after treatment with indicated concentrations of DNA-PK inhibitor (NU-7441) in DLD1 BRCA2 −/− or DLD1 Parental cells. Data represent mean. n = 1 experiment with triplicate for each condition, ± s.e.m. b. Bar plots showing percentage of colonies relative to control after siRNA transfection with either siControl or siPolθ in DLD1 BRCA2 −/− or DLD1 Parental cells (top), in MDA 436 BRCA1 mut or MDA 231 cells (bottom). Percentage of colonies are normalized to non-targeting siRNA (siControl = 100). Data represent mean. n = 1 experiment with triplicate for each condition, ± s.e.m. Colony images are on the right.
a. Structural comparison of Polλ bound to a microhomology-mediated DNA synapse and a single nucleotide gap (PDB id 7UN7). Differences (0–4.2 Å) in backbone Cα positioning are displayed as a heatmap colored from blue (0 Å) to white (0.5 Å) to red (1+Å) mapped onto the structure of the double strand break bound pol λ in cartoon representation. b. Overlay of the microhomology substrate containing gaps in template and primer strands with a single nucleotide gap substrate (PDB id 7UN7, transparent gray). Incoming TTP (green) or dUMPNPP (transparent gray) and downstream (magenta), primer (cyan), template (purple) strands are shown in stick representation. The orange spheres are active site metal ions and the purple sphere is a sodium atom. c. Template strand interactions in DSB bound Polλ. Template strand (magenta) and sidechains (yellow) are shown in stick representation. Key interactions are shown with black (side chains) or green (water) dashes. Waters are shown as blue spheres. Inset. Comparison of template strand positioning and gap marginal nucleotide interactions in structures of Polλ with a nick in the template strand (white transparent sticks, PDB id 7M0D31) and a gap in the same position (yellow sidechains, purple DNA). d. Differences in downstream 5′ phosphate coordination overlaid with the structure of a single nucleotide gap (PDB id 7UN7, transparent gray). e. Lyase domain and downstream primer shift compared to the structure with a single nucleotide gap (PDB id 7UN7). Protein backbone and DNA are shown in magenta cartoon and stick representation, respectively. f. Metal coordination in the active site. Shown is an overlay with a structure of Polλ bound to a single nucleotide gap and a non-hydrolyzable nucleotide (PDB id 7UN7, transparent gray).
Data related to Fig. 2d,g and h.
Data related to Fig. 3b–h.
prizm file in zip format of data related to Fig. 3b–h used for creating bar plots. Pdf file has all uncropped western images.
All uncropped western images.
Data related to Extended Data Fig. 3a,c and e.
Pdf file has all uncropped western images.
Data related to Extended Data Fig. 4a,b.
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Chandramouly, G., Jamsen, J., Borisonnik, N. et al. Polλ promotes microhomology-mediated end-joining. Nat Struct Mol Biol 30, 107–114 (2023). https://doi.org/10.1038/s41594-022-00895-4
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