Two-ended recombination at a Flp-nickase-broken replication fork.

Collision of a replication fork with a DNA nick is thought to generate a one-ended break, fostering genomic instability. Collision of the opposing converging fork with the nick could, in principle, form a second DNA end, enabling conservative repair by homologous recombination (HR). To study mechanisms of nickase-induced HR, we developed the Flp recombinase "step arrest" nickase in mammalian cells. Flp-nickase-induced HR entails two-ended, BRCA2/RAD51-dependent short tract gene conversion (STGC), BRCA2/RAD51-independent long tract gene conversion, and discoordinated two-ended invasions. HR induced by a replication-independent break and by the Flp-nickase differ in their dependence on BRCA1 . To determine the origin of the second DNA end during Flp-nickase-induced STGC, we blocked the opposing fork using a site-specific Tus/ Ter replication fork barrier. Flp-nickase-induced STGC remained robust and two-ended. Thus, collision of a single replication fork with a Flp-nick can trigger two-ended HR, possibly reflecting replicative bypass of lagging strand nicks. This response may limit genomic instability during replication of a nicked DNA template.

A modified CUT&RUN-seq technique for qPCR analysis of chromatin-protein interactions.

Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) even with optimization may give low signal-to-background ratio and spatial resolution. Here, we adapted Cleavage Under Targets and Release Using Nuclease (CUT&RUN) (originally developed by the Henikoff group) to develop CUT&RUN-qPCR. By studying the recruitment of selected proteins (but amenable to other proteins), we find that CUT&RUN-qPCR is more sensitive and gives better spatial resolution than ChIP-qPCR. For complete details on the use and execution of this protocol, please refer to Skene et al. (2018) and Skene and Henikoff (2017).

The structure-specific endonuclease complex SLX4-XPF regulates Tus-Ter-induced homologous recombination.

Vertebrate replication forks arrested at interstrand DNA cross-links (ICLs) engage the Fanconi anemia pathway to incise arrested forks, 'unhooking' the ICL and forming a double strand break (DSB) that is repaired by homologous recombination (HR). The FANCP product, SLX4, in complex with the XPF (also known as FANCQ or ERCC4)-ERCC1 endonuclease, mediates ICL unhooking. Whether this mechanism operates at replication fork barriers other than ICLs is unknown. Here, we study the role of mouse SLX4 in HR triggered by a site-specific chromosomal DNA-protein replication fork barrier formed by the Escherichia coli-derived Tus-Ter complex. We show that SLX4-XPF is required for Tus-Ter-induced HR but not for error-free HR induced by a replication-independent DSB. We additionally uncover a role for SLX4-XPF in DSB-induced long-tract gene conversion, an error-prone HR pathway related to break-induced replication. Notably, Slx4 and Xpf mutants that are defective for Tus-Ter-induced HR are hypersensitive to ICLs and also to the DNA-protein cross-linking agents 5-aza-2'-deoxycytidine and zebularine. Collectively, these findings show that SLX4-XPF can process DNA-protein fork barriers for HR and that the Tus-Ter system recapitulates this process.

Exploiting CRISPR/Cas9 to engineer precise segmental deletions in mouse embryonic stem cells.

In this protocol, we use CRISPR/Cas9 to generate large deletions of the entire coding region of a gene of interest, generating a hemizygous cell line. Next, we systematically engineer precise in-frame deletions within the intact wild-type allele, facilitating study of multi-domain proteins. The optimized protocol described here allows us to rapidly screen for effective sgRNA pairs and to engineer either an in-frame deletion or a frameshift mutation in high frequencies in mouse embryonic stem cells. For complete details on the use and execution of this protocol, please refer to Panday et al. (2021).

The Protexin complex counters resection on stalled forks to promote homologous recombination and crosslink repair.

Protection of stalled replication forks is critical to genomic stability. Using genetic and proteomic analyses, we discovered the Protexin complex containing the ssDNA binding protein SCAI and the DNA polymerase REV3. Protexin is required specifically for protecting forks stalled by nucleotide depletion, fork barriers, fragile sites, and DNA inter-strand crosslinks (ICLs), where it promotes homologous recombination and repair. Protexin loss leads to ssDNA accumulation and profound genomic instability in response to ICLs. Protexin interacts with RNA POL2, and both oppose EXO1's resection of DNA on forks remodeled by the FANCM translocase activity. This pathway acts independently of BRCA/RAD51-mediated fork stabilization, and cells with BRCA2 mutations were dependent on SCAI for survival. These data suggest that Protexin and its associated factors establish a new fork protection pathway that counteracts fork resection in part through a REV3 polymerase-dependent resynthesis mechanism of excised DNA, particularly at ICL stalled forks.

Recombination and restart at blocked replication forks.

Replication fork stalling occurs when the replisome encounters a barrier to normal fork progression. Replisome stalling events are common during scheduled DNA synthesis, but vary in their severity. At one extreme, a lesion may induce only temporary pausing of a DNA polymerase; at the other, it may present a near-absolute barrier to the replicative helicase and effectively block fork progression. Many alternative pathways have evolved to respond to these different types of replication stress. Among these, the homologous recombination (HR) pathway plays an important role, protecting the stalled fork and processing it for repair. Here, we review recent advances in our understanding of how blocked replication forks in vertebrate cells can be processed for recombination and for replication restart.

Lamin B1 sequesters 53BP1 to control its recruitment to DNA damage.

Double-strand breaks (DSBs) are harmful lesions and a major cause of genome instability. Studies have suggested a link between the nuclear envelope and the DNA damage response. Here, we show that lamin B1, a major component of the nuclear envelope, interacts directly with 53BP1 protein, which plays a pivotal role in the DSB repair. This interaction is dissociated after DNA damage. Lamin B1 overexpression impedes 53BP1 recruitment to DNA damage sites and leads to a persistence of DNA damage, a defect in nonhomologous end joining and an increased sensitivity to DSBs. The identification of interactions domains between lamin B1 and 53BP1 allows us to demonstrate that the defect of 53BP1 recruitment and the DSB persistence upon lamin B1 overexpression are due to sequestration of 53BP1 by lamin B1. This study highlights lamin B1 as a factor controlling the recruitment of 53BP1 to DNA damage sites upon injury.

FANCM regulates repair pathway choice at stalled replication forks.

Repair pathway "choice" at stalled mammalian replication forks is an important determinant of genome stability; however, the underlying mechanisms are poorly understood. FANCM encodes a multi-domain scaffolding and motor protein that interacts with several distinct repair protein complexes at stalled forks. Here, we use defined mutations engineered within endogenous Fancm in mouse embryonic stem cells to study how Fancm regulates stalled fork repair. We find that distinct FANCM repair functions are enacted by molecularly separable scaffolding domains. These findings define FANCM as a key mediator of repair pathway choice at stalled replication forks and reveal its molecular mechanism. Notably, mutations that inactivate FANCM ATPase function disable all its repair functions and "trap" FANCM at stalled forks. We find that Brca1 hypomorphic mutants are synthetic lethal with Fancm null or Fancm ATPase-defective mutants. The ATPase function of FANCM may therefore represent a promising "druggable" target for therapy of BRCA1-linked cancer.

Measurement of Homologous Recombination at Stalled Mammalian Replication Forks.

Site-specific replication fork barriers (RFBs) have proven valuable tools for studying mechanisms of repair at sites of replication fork stalling in prokaryotes and yeasts. We adapted the Escherichia coli Tus-Ter RFB for use in mammalian cells and used it to trigger site-specific replication fork stalling and homologous recombination (HR) at a defined chromosomal locus in mammalian cells. By comparing HR responses induced at the Tus-Ter RFB with those induced by a site-specific double-strand break (DSB), we have begun to uncover how the mechanisms of mammalian stalled fork repair differ from those underlying the repair of a replication-independent DSB. Here, we outline how to transiently express the Tus protein in mES cells, how to use flow cytometry to score conservative and aberrant repair outcomes, and how to quantify distinct repair outcomes in response to replication fork stalling at the inducible Tus-Ter chromosomal RFB.

Inactivation of the Prolyl Isomerase Pin1 Sensitizes BRCA1-Proficient Breast Cancer to PARP Inhibition.

PARP inhibitor monotherapies are effective to treat patients with breast, ovary, prostate, and pancreatic cancer with BRCA1 mutations, but not to the much more frequent BRCA wild-type cancers. Searching for strategies that would extend the use of PARP inhibitors to BRCA1-proficient tumors, we found that the stability of BRCA1 protein following ionizing radiation (IR) is maintained by postphosphorylational prolyl-isomerization adjacent to Ser1191 of BRCA1, catalyzed by prolyl-isomerase Pin1. Extinction of Pin1 decreased homologous recombination (HR) to the level of BRCA1-deficient cells. Pin1 stabilizes BRCA1 by preventing ubiquitination of Lys1037 of BRCA1. Loss of Pin1, or introduction of a BRCA1-mutant refractory to Pin1 binding, decreased the ability of BRCA1 to localize to repair foci and augmented IR-induced DNA damage. In vitro growth of HR-proficient breast, prostate, and pancreatic cancer cells were modestly repressed by olaparib or Pin1 inhibition using all-trans retinoic acid (ATRA), while combination treatment resulted in near-complete block of cell proliferation. In MDA-MB-231 xenografts and triple-negative breast cancer patient-derived xenografts, either loss of Pin1 or ATRA treatment reduced BRCA1 expression and sensitized breast tumors to olaparib. Together, our study reveals that Pin1 inhibition, with clinical widely used ATRA, acts as an effective HR disrupter that sensitizes BRCA1-proficient tumors to PARP inhibition. SIGNIFICANCE: PARP inhibitors have been limited to treat homologous recombination-deficient tumors. All-trans retinoic acid, by inhibiting Pin1 and destabilizing BRCA1, extends benefit of PARP inhibitors to patients with homologous recombination-proficient tumors.See related commentary by Cai, p. 2977.

DNA double-strand break repair-pathway choice in somatic mammalian cells.

The major pathways of DNA double-strand break (DSB) repair are crucial for maintaining genomic stability. However, if deployed in an inappropriate cellular context, these same repair functions can mediate chromosome rearrangements that underlie various human diseases, ranging from developmental disorders to cancer. The two major mechanisms of DSB repair in mammalian cells are non-homologous end joining (NHEJ) and homologous recombination. In this Review, we consider DSB repair-pathway choice in somatic mammalian cells as a series of 'decision trees', and explore how defective pathway choice can lead to genomic instability. Stalled, collapsed or broken DNA replication forks present a distinctive challenge to the DSB repair system. Emerging evidence suggests that the 'rules' governing repair-pathway choice at stalled replication forks differ from those at replication-independent DSBs.

The Tandem Duplicator Phenotype Is a Prevalent Genome-Wide Cancer Configuration Driven by Distinct Gene Mutations.

The tandem duplicator phenotype (TDP) is a genome-wide instability configuration primarily observed in breast, ovarian, and endometrial carcinomas. Here, we stratify TDP tumors by classifying their tandem duplications (TDs) into three span intervals, with modal values of 11 kb, 231 kb, and 1.7 Mb, respectively. TDPs with ∼11 kb TDs feature loss of TP53 and BRCA1. TDPs with ∼231 kb and ∼1.7 Mb TDs associate with CCNE1 pathway activation and CDK12 disruptions, respectively. We demonstrate that p53 and BRCA1 conjoint abrogation drives TDP induction by generating short-span TDP mammary tumors in genetically modified mice lacking them. Lastly, we show how TDs in TDP tumors disrupt heterogeneous combinations of tumor suppressors and chromatin topologically associating domains while duplicating oncogenes and super-enhancers.

Rad51 recruitment and exclusion of non-homologous end joining during homologous recombination at a Tus/Ter mammalian replication fork barrier.

Classical non-homologous end joining (C-NHEJ) and homologous recombination (HR) compete to repair mammalian chromosomal double strand breaks (DSBs). However, C-NHEJ has no impact on HR induced by DNA nicking enzymes. In this case, the replication fork is thought to convert the DNA nick into a one-ended DSB, which lacks a readily available partner for C-NHEJ. Whether C-NHEJ competes with HR at a non-enzymatic mammalian replication fork barrier (RFB) remains unknown. We previously showed that conservative "short tract" gene conversion (STGC) induced by a chromosomal Tus/Ter RFB is a product of bidirectional replication fork stalling. This finding raises the possibility that Tus/Ter-induced STGC proceeds via a two-ended DSB intermediate. If so, Tus/Ter-induced STGC might be subject to competition by C-NHEJ. However, in contrast to the DSB response, where genetic ablation of C-NHEJ stimulates HR, we report here that Tus/Ter-induced HR is unaffected by deletion of either of two C-NHEJ genes, Xrcc4 or Ku70. These results show that Tus/Ter-induced HR does not entail the formation of a two-ended DSB to which C-NHEJ has competitive access. We found no evidence that the alternative end-joining factor, DNA polymerase θ, competes with Tus/Ter-induced HR. We used chromatin-immunoprecipitation to compare Rad51 recruitment to a Tus/Ter RFB and to a neighboring site-specific DSB. Rad51 accumulation at Tus/Ter was more intense and more sustained than at a DSB. In contrast to the DSB response, Rad51 accumulation at Tus/Ter was restricted to within a few hundred base pairs of the RFB. Taken together, these findings suggest that the major DNA structures that bind Rad51 at a Tus/Ter RFB are not conventional DSBs. We propose that Rad51 acts as an "early responder" at stalled forks, binding single stranded daughter strand gaps on the arrested lagging strand, and that Rad51-mediated fork remodeling generates HR intermediates that are incapable of Ku binding and therefore invisible to the C-NHEJ machinery.

Mechanism of tandem duplication formation in BRCA1-mutant cells.

Small, approximately 10-kilobase microhomology-mediated tandem duplications are abundant in the genomes of BRCA1-linked but not BRCA2-linked breast cancer. Here we define the mechanism underlying this rearrangement signature. We show that, in primary mammalian cells, BRCA1, but not BRCA2, suppresses the formation of tandem duplications at a site-specific chromosomal replication fork barrier imposed by the binding of Tus proteins to an array of Ter sites. BRCA1 has no equivalent role at chromosomal double-stranded DNA breaks, indicating that tandem duplications form specifically at stalled forks. Tandem duplications in BRCA1 mutant cells arise by a replication restart-bypass mechanism terminated by end joining or by microhomology-mediated template switching, the latter forming complex tandem duplication breakpoints. Solitary DNA ends form directly at Tus-Ter, implicating misrepair of these lesions in tandem duplication formation. Furthermore, BRCA1 inactivation is strongly associated with ~10 kilobase tandem duplications in ovarian cancer. This tandem duplicator phenotype may be a general signature of BRCA1-deficient cancer.

FANCJ helicase controls the balance between short- and long-tract gene conversions between sister chromatids.

The FANCJ DNA helicase is linked to hereditary breast and ovarian cancers as well as bone marrow failure disorder Fanconi anemia (FA). Although FANCJ has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR), the molecular mechanism underlying the tumor suppressor functions of FANCJ remains obscure. Here, we demonstrate that FANCJ deficient human and hamster cells exhibit reduction in the overall gene conversions in response to a site-specific chromosomal DSB induced by I-SceI endonuclease. Strikingly, the gene conversion events were biased in favour of long-tract gene conversions in FANCJ depleted cells. The fine regulation of short- (STGC) and long-tract gene conversions (LTGC) by FANCJ was dependent on its interaction with BRCA1 tumor suppressor. Notably, helicase activity of FANCJ was essential for controlling the overall HR and in terminating the extended repair synthesis during sister chromatid recombination (SCR). Moreover, cells expressing FANCJ pathological mutants exhibited defective SCR with an increased frequency of LTGC. These data unravel the novel function of FANCJ helicase in regulating SCR and SCR associated gene amplification/duplications and imply that these functions of FANCJ are crucial for the genome maintenance and tumor suppression.

Global increase in replication fork speed during a p57KIP2-regulated erythroid cell fate switch.

Cell cycle regulators are increasingly implicated in cell fate decisions, such as the acquisition or loss of pluripotency and self-renewal potential. The cell cycle mechanisms that regulate these cell fate decisions are largely unknown. We studied an S phase-dependent cell fate switch, in which murine early erythroid progenitors transition in vivo from a self-renewal state into a phase of active erythroid gene transcription and concurrent maturational cell divisions. We found that progenitors are dependent on p57KIP2-mediated slowing of replication forks for self-renewal, a novel function for cyclin-dependent kinase inhibitors. The switch to differentiation entails rapid down-regulation of p57KIP2 with a consequent global increase in replication fork speed and an abruptly shorter S phase. Our work suggests that cell cycles with specialized global DNA replication dynamics are integral to the maintenance of specific cell states and to cell fate decisions.

DEK is required for homologous recombination repair of DNA breaks.

DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition.

Correction: Complex Breakpoints and Template Switching Associated with Non-canonical Termination of Homologous Recombination in Mammalian Cells.

[This corrects the article DOI: 10.1371/journal.pgen.1006410.].

Complex Breakpoints and Template Switching Associated with Non-canonical Termination of Homologous Recombination in Mammalian Cells.

A proportion of homologous recombination (HR) events in mammalian cells resolve by "long tract" gene conversion, reflecting copying of several kilobases from the donor sister chromatid prior to termination. Cells lacking the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, or certain other HR-defective cells, reveal a bias in favor of long tract gene conversion, suggesting that this aberrant HR outcome might be connected with genomic instability. If termination of gene conversion occurs in regions lacking homology with the second end of the break, the normal mechanism of HR termination by annealing (i.e., homologous pairing) is not available and termination must occur by as yet poorly defined non-canonical mechanisms. Here we use a previously described HR reporter to analyze mechanisms of non-canonical termination of long tract gene conversion in mammalian cells. We find that non-canonical HR termination can occur in the absence of the classical non-homologous end joining gene XRCC4. We observe obligatory use of microhomology (MH)-mediated end joining and/or nucleotide addition during rejoining with the second end of the break. Notably, non-canonical HR termination is associated with complex breakpoints. We identify roles for homology-mediated template switching and, potentially, MH-mediated template switching/microhomology-mediated break-induced replication, in the formation of complex breakpoints at sites of non-canonical HR termination. This work identifies non-canonical HR termination as a potential contributor to genomic instability and to the formation of complex breakpoints in cancer.