Although a defect in the DNA polymerase POLQ leads to ionizing radiation sensitivity in mammalian cells, the relevant enzymatic pathway has not been identified. Here we define the specific mechanism by which POLQ restricts harmful DNA instability. Our experiments show that Polq-null murine cells are selectively hypersensitive to DNA strand breaking agents, and that damage resistance requires the DNA polymerase activity of POLQ. Using a DNA break end joining assay in cells, we monitored repair of DNA ends with long 3′ single-stranded overhangs. End joining events retaining much of the overhang were dependent on POLQ, and independent of Ku70. To analyze the repair function in more detail, we examined immunoglobulin class switch joining between DNA segments in antibody genes. POLQ participates in end joining of a DNA break during immunoglobulin class-switching, producing insertions of base pairs at the joins with homology to IgH switch-region sequences. Biochemical experiments with purified human POLQ protein revealed the mechanism generating the insertions during DNA end joining, relying on the unique ability of POLQ to extend DNA from minimally paired primers. DNA breaks at the IgH locus can sometimes join with breaks in Myc, creating a chromosome translocation. We found a marked increase in Myc/IgH translocations in Polq-defective mice, showing that POLQ suppresses genomic instability and genome rearrangements originating at DNA double-strand breaks. This work clearly defines a role and mechanism for mammalian POLQ in an alternative end joining pathway that suppresses the formation of chromosomal translocations. Our findings depart from the prevailing view that alternative end joining processes are generically translocation-prone. The reason for the hypersensitivity of POLQ-defective mammalian cells to ionizing radiation has been elusive. Here we show that POLQ-defective mammalian cells are selectively susceptible to double-strand breaks in DNA. We present experiments in mammalian cells showing that a specific double-strand break repair pathway is POLQ-dependent. To analyze the repair function in more detail, we examined class switch joining between DNA segments in antibody genes. Insertions of DNA bases are sometimes found at the joins between such segments, but the origin of these insertions has been mysterious. We show that this class of insertion joins during immunoglobulin class-switching is entirely POLQ-dependent. In experiments with purified human POLQ protein, we found a novel biochemical mechanism explaining the formation of the insertions. POLQ has a unique biochemical ability to extend DNA with minimal base pairing. Finally, we examined the biological consequences for chromosome stability. Unexpectedly, the Burkitt lymphoma translocation (a major cancer-associated genome instability) is enhanced in the absence of POLQ. This alters the current view about the action of DNA end joining in mammalian cells, revealing that a POLQ-dependent DNA repair pathway combats potentially damaging chromosome translocations.

To exploit vulnerabilities of tumors, it is urgent to identify associated defects in genome maintenance. One unsolved problem is the mechanism of regulation of DNA double‐strand break repair by REV7 in complex with 53BP1 and RIF1, and its influence on repair pathway choice between homologous recombination and non‐homologous end‐joining. We searched for REV7‐associated factors in human cells and found FAM35A, a previously unstudied protein with an unstructured N‐terminal region and a C‐terminal region harboring three OB‐fold domains similar to single‐stranded DNA‐binding protein RPA, as novel interactor of REV7/RIF1/53BP1. FAM35A re‐localized in damaged cell nuclei, and its knockdown caused sensitivity to DNA‐damaging agents. In a BRCA1‐mutant cell line, however, depletion of FAM35A increased resistance to camptothecin, suggesting that FAM35A participates in processing of DNA ends to allow more efficient DNA repair. We found FAM35A absent in one widely used BRCA1‐mutant cancer cell line (HCC1937) with anomalous resistance to PARP inhibitors. A survey of FAM35A alterations revealed that the gene is altered at the highest frequency in prostate cancers (up to 13%) and significantly less expressed in metastatic cases, revealing promise for FAM35A as a therapeutically relevant cancer marker.

When a replicative DNA polymerase (Pol) is stalled by damaged DNA, a “polymerase switch” recruits specialized translesion synthesis (TLS) DNA polymerase(s) to sites of damage. Mammalian cells have several TLS DNA polymerases, including the four Y‐family enzymes (Polη, Polι, Polκ and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Polη, Polι, and Polκ and therefore appears to play a central role during TLS in vivo. Here we have investigated the molecular basis for interactions between REV1 and Polκ. We have identified novel REV1‐interacting regions (RIRs) present in Polκ, Polι and Polη. Within the RIRs, the presence of two consecutive phenylalanines (FF) is essential for REV1‐binding. The consensus sequence for REV1‐binding is denoted by x‐x‐x‐F‐F‐y‐y‐y‐y (x, no specific residue and y, no specific residue but not proline). Our results identify structural requirements that are necessary for FF‐flanking residues to confer interactions with REV1. A Polκ mutant lacking REV1‐binding activity did not complement the genotoxin‐sensitivity of Polk‐null mouse embryonic fibroblast cells, thereby demonstrating that the REV1‐interaction is essential for Polκ function in vivo.

Mammalian HELQ is a 3′–5′ DNA helicase with strand displacement activity. Here we show that HELQ participates in a pathway of resistance to DNA interstrand crosslinks (ICLs). Genetic disruption ofHELQin human cells enhances cellular sensitivity and chromosome radial formation by the ICL-inducing agent mitomycin C (MMC). A significant fraction of MMC sensitivity is independent of the Fanconi anaemia pathway. Sister chromatid exchange frequency and sensitivity to UV radiation or topoisomerase inhibitors is unaltered. Proteomic analysis reveals that HELQ is associated with the RAD51 paralogs RAD51B/C/D and XRCC2, and with the DNA damage-responsive kinase ATR. After treatment with MMC, reduced phosphorylation of the ATR substrate CHK1 occurs inHELQ-knockout cells, and accumulation of G₂/M cells is reduced. The results indicate that HELQ operates in an arm of DNA repair and signalling in response to ICL. Further, the association with RAD51 paralogs suggests HELQ as a candidate ovarian cancer gene.