Sequential requirements for distinct Polθ domains during theta-mediated end joining.

DNA polymerase θ (Polθ) plays a central role in a DNA double-strand break repair pathway termed theta-mediated end joining (TMEJ). TMEJ functions by pairing short-sequence "microhomologies" (MHs) in single-stranded DNA at each end of a break and subsequently initiating DNA synthesis. It is not known how the Polθ helicase domain (HD) and polymerase domain (PD) operate to bring together MHs and facilitate repair. To resolve these transient processes in real time, we utilized in vitro single-molecule FRET approaches and biochemical analyses. We find that the Polθ-HD mediates the initial capture of two ssDNA strands, bringing them in close proximity. The Polθ-PD binds and stabilizes pre-annealed MHs to form a synaptic complex (SC) and initiate repair synthesis. Individual synthesis reactions show that Polθ is inherently non-processive, accounting for complex mutational patterns during TMEJ. Binding of Polθ-PD to stem-loop-forming sequences can substantially limit synapsis, depending on the available dNTPs and sequence context.

Errol Friedberg: A life in writing.

Errol Clive Friedberg, who died at the end of March 2023, was the first Editor-in-Chief of the journal DNA Repair. He was an influential DNA repair scientist, a synthesizer of ideas, and an accomplished historian. In addition to the research accomplishments of his laboratory groups, Errol Friedberg provided enormous service to the DNA repair community though organizing major conferences, journal editing, and writing. His many books include texts about DNA repair, histories of the field, and biographies of several pioneers of molecular biology.

Genome Protection by DNA Polymerase θ.

DNA polymerase θ (Pol θ) is a DNA repair enzyme widely conserved in animals and plants. Pol θ uses short DNA sequence homologies to initiate repair of double-strand breaks by theta-mediated end joining. The DNA polymerase domain of Pol θ is at the C terminus and is connected to an N-terminal DNA helicase-like domain by a central linker. Pol θ is crucial for maintenance of damaged genomes during development, protects DNA against extensive deletions, and limits loss of heterozygosity. The cost of using Pol θ for genome protection is that a few nucleotides are usually deleted or added at the repair site. Inactivation of Pol θ often enhances the sensitivity of cells to DNA strand-breaking chemicals and radiation. Since some homologous recombination-defective cancers depend on Pol θ for growth, inhibitors of Pol θ may be useful in treating such tumors.

Probing the structure and function of polymerase θ helicase-like domain.

DNA Polymerase θ is the key actuator of the recently identified double-strand break repair pathway, theta-mediated end joining (TMEJ). It is the only known polymerase to have a 3-domain architecture containing an independently functional family A DNA polymerase tethered by a long central region to an N-terminal helicase-like domain (HLD). Full-length polymerase θ and the isolated HLD hydrolyze ATP in the presence of DNA, but no processive DNA duplex unwinding has been observed. Based on sequence and structure conservation, the HLD is classified as a member of helicase superfamily II and, more specifically, the Ski2-like family. The specific subdomain composition and organization most closely resemble that of archaeal DNA repair helicases Hel308 and Hjm. The underlying structural basis as to why the HLD is not able to processively unwind duplex DNA, despite its similarity to bona fide helicases, remains elusive. Activities of the HLD include ATP hydrolysis, protein displacement, and annealing of complementary DNA. These observations have led to speculation about the role of the HLD within the context of double-strand break repair via TMEJ, such as removal of single-stranded DNA binding proteins like RPA and RAD51 and microhomology alignment. This review summarizes the structural classification and organization of the polymerase θ HLD and its homologs and explores emerging data on its biochemical activities. We conclude with a simple, speculative model for the HLD's role in TMEJ.

POLθ-mediated end joining is restricted by RAD52 and BRCA2 until the onset of mitosis.

BRCA2-mutant cells are defective in homologous recombination, making them vulnerable to the inactivation of other pathways for the repair of DNA double-strand breaks (DSBs). This concept can be clinically exploited but is currently limited due to insufficient knowledge about how DSBs are repaired in the absence of BRCA2. We show that DNA polymerase θ (POLθ)-mediated end joining (TMEJ) repairs DSBs arising during the S phase in BRCA2-deficient cells only after the onset of the ensuing mitosis. This process is regulated by RAD52, whose loss causes the premature usage of TMEJ and the formation of chromosomal fusions. Purified RAD52 and BRCA2 proteins both block the DNA polymerase function of POLθ, suggesting a mechanism explaining their synthetic lethal relationships. We propose that the delay of TMEJ until mitosis ensures the conversion of originally one-ended DSBs into two-ended DSBs. Mitotic chromatin condensation might further serve to juxtapose correct break ends and limit chromosomal fusions.

DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability.

The DNA polymerase zeta (Polζ) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polζ, is also essential in mouse embryonic development and cell proliferation for reasons that remain incompletely understood. In this study, we reveal that REV3L protein interacts with heterochromatin components including repressive histone marks and localizes in pericentromeric regions through direct interaction with HP1 dimer. We demonstrate that Polζ/REV3L ensures progression of replication forks through difficult-to-replicate pericentromeric heterochromatin, thereby preventing spontaneous chromosome break formation. We also find that Rev3l-deficient cells are compromised in the repair of heterochromatin-associated double-stranded breaks, eliciting deletions in late-replicating regions. Lack of REV3L leads to further consequences that may be ascribed to heterochromatin replication and repair-associated functions of Polζ, with a disruption of the temporal replication program at specific loci. This is correlated with changes in epigenetic landscape and transcriptional control of developmentally regulated genes. These results reveal a new function of Polζ in preventing chromosome instability during replication of heterochromatic regions.

Regulating Polθ in Breast Cancer.

DNA polymerase θ, a protein encoded by the POLQ gene, is the defining factor for the DNA double-strand break repair pathway known as theta-mediated end-joining (TMEJ). Some cancers depend on TMEJ for survival and tumor growth. TMEJ might be useful as a biomarker to guide patient treatment and is now an active target for drug development, making it critical to understand how it is regulated in cells. In a recent article, Prodhomme and colleagues provide the first identification of a transcription regulator of POLQ expression and TMEJ activity: the transcription factor, ZEB1.See related article by Prodhomme et al., p. 1595.

Disruption of DNA polymerase ζ engages an innate immune response.

In mammalian cells, specialized DNA polymerase ζ (pol ζ) contributes to genomic stability during normal DNA replication. Disruption of the catalytic subunit Rev3l is toxic and results in constitutive chromosome damage, including micronuclei. As manifestations of this genomic stress are unknown, we examined the transcriptome of pol ζ-defective cells by RNA sequencing (RNA-seq). Expression of 1,117 transcripts is altered by ≥4-fold in Rev3l-disrupted cells, with a pattern consistent with an induction of an innate immune response. Increased expression of interferon-stimulated genes at the mRNA and protein levels in pol ζ-defective cells is driven by the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-signaling partner stimulator of interferon genes (STING) pathway. Expression of key interferon-stimulated chemokines is elevated in basal epithelial mouse skin cells with a disruption of Rev3l. These results indicate that the disruption of pol ζ may simultaneously increase sensitivity to genotoxins and potentially engage parts of the innate immune response, which could add an additional benefit to targeting pol ζ in cancer therapies.

Human DNA polymerase θ harbors DNA end-trimming activity critical for DNA repair.

Cancers with hereditary defects in homologous recombination rely on DNA polymerase θ (pol θ) for repair of DNA double-strand breaks. During end joining, pol θ aligns microhomology tracts internal to 5'-resected broken ends. An unidentified nuclease trims the 3' ends before synthesis can occur. Here we report that a nuclease activity, which differs from the proofreading activity often associated with DNA polymerases, is intrinsic to the polymerase domain of pol θ. Like the DNA synthesis activity, the nuclease activity requires conserved metal-binding residues, metal ions, and dNTPs and is inhibited by ddNTPs or chain-terminated DNA. Our data indicate that pol θ repurposes metal ions in the polymerase active site for endonucleolytic cleavage and that the polymerase-active and end-trimming conformations of the enzyme are distinct. We reveal a nimble strategy of substrate processing that allows pol θ to trim or extend DNA depending on the DNA repair context.

When DNA Polymerases Multitask: Functions Beyond Nucleotidyl Transfer.

DNA polymerases catalyze nucleotidyl transfer, the central reaction in synthesis of DNA polynucleotide chains. They function not only in DNA replication, but also in diverse aspects of DNA repair and recombination. Some DNA polymerases can perform translesion DNA synthesis, facilitating damage tolerance and leading to mutagenesis. In addition to these functions, many DNA polymerases conduct biochemically distinct reactions. This review presents examples of DNA polymerases that carry out nuclease (3'-5' exonuclease, 5' nuclease, or end-trimming nuclease) or lyase (5' dRP lyase) extracurricular activities. The discussion underscores how DNA polymerases have a remarkable ability to manipulate DNA strands, sometimes involving relatively large intramolecular movement.

DNA polymerase ι compensates for Fanconi anemia pathway deficiency by countering DNA replication stress.

Fanconi anemia (FA) is caused by defects in cellular responses to DNA crosslinking damage and replication stress. Given the constant occurrence of endogenous DNA damage and replication fork stress, it is unclear why complete deletion of FA genes does not have a major impact on cell proliferation and germ-line FA patients are able to progress through development well into their adulthood. To identify potential cellular mechanisms that compensate for the FA deficiency, we performed dropout screens in FA mutant cells with a whole genome guide RNA library. This uncovered a comprehensive genome-wide profile of FA pathway synthetic lethality, including POLI and CDK4 As little is known of the cellular function of DNA polymerase iota (Pol ι), we focused on its role in the loss-of-function FA knockout mutants. Loss of both FA pathway function and Pol ι leads to synthetic defects in cell proliferation and cell survival, and an increase in DNA damage accumulation. Furthermore, FA-deficient cells depend on the function of Pol ι to resume replication upon replication fork stalling. Our results reveal a critical role for Pol ι in DNA repair and replication fork restart and suggest Pol ι as a target for therapeutic intervention in malignancies carrying an FA gene mutation.

Defining the mutation signatures of DNA polymerase θ in cancer genomes.

DNA polymerase theta (POLQ)-mediated end joining (TMEJ) is a distinct pathway for mediating DNA double-strand break (DSB) repair. TMEJ is required for the viability of BRCA-mutated cancer cells. It is crucial to identify tumors that rely on POLQ activity for DSB repair, because such tumors are defective in other DSB repair pathways and have predicted sensitivity to POLQ inhibition and to cancer therapies that produce DSBs. We define here the POLQ-associated mutation signatures in human cancers, characterized by short insertions and deletions in a specific range of microhomologies. By analyzing 82 COSMIC (Catalogue of Somatic Mutations in Cancer) signatures, we found that BRCA-mutated cancers with a higher level of POLQ expression have a greatly enhanced representation of the small insertion and deletion signature 6, as well as single base substitution signature 3. Using human cancer cells with disruptions of POLQ, we further show that TMEJ dominates end joining of two separated DSBs (distal EJ). Templated insertions with microhomology are enriched in POLQ-dependent distal EJ. The use of this signature analysis will aid in identifying tumors relying on POLQ activity.

Mechanistic basis for microhomology identification and genome scarring by polymerase theta.

DNA polymerase theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3' single-stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3' terminus and favoring bidirectional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3 to 30 bp of sequence that is identical to flanking DNA ("templated" insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification and shows how it both mitigates limitations implicit in the microhomology requirement and generates distinctive genomic scars associated with pathogenic genome instability.

Genetic determinants of cellular addiction to DNA polymerase theta.

Polymerase theta (Pol θ, gene name Polq) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction. Here we use a CRISPR genetic screen to uncover 140 Polq synthetic lethal (PolqSL) genes, the majority of which were previously unknown. Functional analyses indicate that Pol θ/TMEJ addiction is associated with increased levels of replication-associated DSBs, regardless of the initial source of damage. We further demonstrate that approximately 30% of TCGA breast cancers have genetic alterations in PolqSL genes and exhibit genomic scars of Pol θ/TMEJ hyperactivity, thereby substantially expanding the subset of human cancers for which Pol θ inhibition represents a promising therapeutic strategy.

CNDAC-Induced DNA Double-Strand Breaks Cause Aberrant Mitosis Prior to Cell Death.

Incorporation of the clinically active deoxycytidine analogue 2'-C-cyano-2'-deoxy-1-ß-D-arabino-pentofuranosyl-cytosine (CNDAC) into DNA generates single-strand breaks that are subsequently converted to double-strand breaks (DSB). Here, we investigated the cellular manifestations of these breaks that link these mechanisms to cell death, and we further tested the relevance of DNA repair pathways in protection of cells against CNDAC damage. The present investigations demonstrate that following exposure to CNDAC and a wash into drug-free medium, chromosomal aberrations, DNA strand breaks, and multinucleate cells arose. These portended loss of viability and were dependent upon exposure time, CNDAC concentration, and passage through mitosis. Following a pulse incubation with CNDAC, live cell imaging using GFP-tagged histone H2B as a marker demonstrated a normal rate of progression to mitosis, but a concentration-dependent delay in passage to a second mitosis. Progression through mitosis was also delayed and accompanied by formation of multinucleate cells. CNDAC-treated cells lacking XPF-ERCC1 nuclease function showed a 16-fold increase in chromosome aberrations. Chromosomal damage in Rad51D-mutant cells (homologous recombination repair deficient) were even more severely affected with extensive aberrations. Rodent or human Polq (POLQ) mutant cells, defective in Pol θ-mediated alternative end joining, did not show enhanced cellular sensitivity to CNDAC. These findings are consistent with formation of DSBs in the second S-phase following exposure, resulting in chromosome aberrations, aberrant mitoses, and subsequent apoptosis.

DNA polymerase ζ in DNA replication and repair.

Here, we survey the diverse functions of DNA polymerase ζ (pol ζ) in eukaryotes. In mammalian cells, REV3L (3130 residues) is the largest catalytic subunit of the DNA polymerases. The orthologous subunit in yeast is Rev3p. Pol ζ also includes REV7 subunits (encoded by Rev7 in yeast and MAD2L2 in mammalian cells) and two subunits shared with the replicative DNA polymerase, pol δ. Pol ζ is used in response to circumstances that stall DNA replication forks in both yeast and mammalian cells. The best-examined situation is translesion synthesis at sites of covalent DNA lesions such as UV radiation-induced photoproducts. We also highlight recent evidence that uncovers various roles of pol ζ that extend beyond translesion synthesis. For instance, pol ζ is also employed when the replisome operates sub-optimally or at difficult-to-replicate DNA sequences. Pol ζ also participates in repair by microhomology mediated break-induced replication. A rev3 deletion is tolerated in yeast but Rev3l disruption results in embryonic lethality in mice. Inactivation of mammalian Rev3l results in genomic instability and invokes cell death and senescence programs. Targeting of pol ζ function may be a useful strategy in cancer therapy, although chromosomal instability associated with pol ζ deficiency must be considered.

DNA polymerase ζ deficiency causes impaired wound healing and stress-induced skin pigmentation.

DNA polymerase ζ (pol ζ) is a specialized enzyme important for DNA damage tolerance, facilitating synthesis past lesions caused by radiation or chemical damage. Here we report that disruption of Rev3l (encoding the catalytic subunit of pol ζ) in mouse epidermis leads to a defect in proliferation that impairs cutaneous wound healing. A striking increase in epidermal skin pigmentation accompanied both wound healing and UV irradiation in these mice. This was a consequence of stress-induced migration of Rev3l-proficient melanocytes to the Rev3l-defective epidermis. This pigmentation corresponded with p53 activation in keratinocytes and was absent in p53-negative areas of the epidermis. Expression of the kit ligand (Kitl) gene, a p53-controlled mediator of keratinocyte to melanocyte signaling, was enhanced during wound healing or following UV irradiation. This study extends the function of pol ζ to the process of proliferation during wound healing. Rev3l-deficient epidermis may be a useful mouse model system for examining communication between damaged keratinocytes and melanocytes, including signaling relevant to human disease.