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).

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.

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.

Tracking break-induced replication shows that it stalls at roadblocks.

Break-induced replication (BIR) repairs one-ended double-strand breaks in DNA similar to those formed by replication collapse or telomere erosion, and it has been implicated in the initiation of genome instability in cancer and other human diseases1,2. Previous studies have defined the enzymes that are required for BIR1-5; however, understanding of initial and extended BIR synthesis, and of how the migrating D-loop proceeds through known replication roadblocks, has been precluded by technical limitations. Here we use a newly developed assay to show that BIR synthesis initiates soon after strand invasion and proceeds more slowly than S-phase replication. Without primase, leading strand synthesis is initiated efficiently, but is unable to proceed beyond 30 kilobases, suggesting that primase is needed for stabilization of the nascent leading strand. DNA synthesis can initiate in the absence of Pif1 or Pol32, but does not proceed efficiently. Interstitial telomeric DNA disrupts and terminates BIR progression, and BIR initiation is suppressed by transcription proportionally to the transcription level. Collisions between BIR and transcription lead to mutagenesis and chromosome rearrangements at levels that exceed instabilities induced by transcription during normal replication. Together, these results provide fundamental insights into the mechanism of BIR and how BIR contributes to genome instability.

Investigation of Break-Induced Replication in Yeast.

Repair of double-strand DNA breaks (DSBs) is important for preserving genomic integrity and stability. Break-induced replication (BIR) is a mechanism aimed to repair one-ended double-strand DNA breaks, similar to those formed by replication fork collapse or by telomere erosion. Unlike S-phase replication, BIR is carried out by a migrating DNA bubble and is associated with conservative inheritance of newly synthesized DNA. This unusual DNA synthesis leads to high level of mutagenesis and chromosomal rearrangements during BIR. Here, we focus on several genetic and molecular methods to investigate BIR using our system in yeast Saccharomyces cerevisiae where BIR is initiated by a site-specific DNA break, and the repair involves two copies of chromosome III.

Repair of base damage within break-induced replication intermediates promotes kataegis associated with chromosome rearrangements.

Break induced replication (BIR) is a double strand break repair pathway that can promote genetic instabilities similar to those observed in cancer. Instead of a replication fork, BIR is driven by a migration bubble where asynchronous synthesis between leading and lagging strands leads to accumulation of single-stranded DNA (ssDNA) that promotes mutation. However, the details of the mechanism of mutagenesis, including the identity of the participating proteins, remain unknown. Using yeast as a model, we demonstrate that mutagenic ssDNA is formed at multiple positions along the BIR track and that Pol ζ is responsible for the majority of both spontaneous and damage-induced base substitutions during BIR. We also report that BIR creates a potent substrate for APOBEC3A (A3A) cytidine deaminase that can promote formation of mutation clusters along the entire track of BIR. Finally, we demonstrate that uracil glycosylase initiates the bypass of DNA damage induced by A3A in the context of BIR without formation of base substitutions, but instead this pathway frequently leads to chromosomal rearrangements. Together, the expression of A3A during BIR in yeast recapitulates the main features of APOBEC-induced kataegis in human cancers, suggesting that BIR might represent an important source of these hyper-mutagenic events.

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.

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways.

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

Investigation of Break-Induced Replication in Yeast.

Break-induced replication (BIR) is an important mechanism aimed to repair one-ended double-strand DNA breaks. BIR is initiated by invasion of a broken DNA end into a homologous template followed by DNA synthesis that can proceed for hundreds of kilobases to the end of the chromosome. Unlike S-phase replication, BIR is carried out by a migrating DNA bubble and is associated with conservative inheritance of newly synthesized DNA. The unusual mode of DNA synthesis during BIR leads to an increased level of genetic instabilities including increased mutagenesis and chromosomal rearrangements. Here, we describe our experimental system in yeast Saccharomyces cerevisiae where BIR is initiated by a site-specific DNA break and where the repair involves two copies of chromosome III. This system allows investigation of BIR using genetic and molecular biology approaches, and can be used for characterization of the BIR mechanism, roles of individual proteins in BIR, and for the analysis of genetic instabilities associated with BIR.

Break-induced replication promotes formation of lethal joint molecules dissolved by Srs2.

Break-induced replication (BIR) is a DNA double-strand break repair pathway that leads to genomic instabilities similar to those observed in cancer. BIR proceeds by a migrating bubble where asynchrony between leading and lagging strand synthesis leads to accumulation of long single-stranded DNA (ssDNA). It remains unknown how this ssDNA is prevented from unscheduled pairing with the template, which can lead to genomic instability. Here, we propose that uncontrolled Rad51 binding to this ssDNA promotes formation of toxic joint molecules that are counteracted by Srs2. First, Srs2 dislodges Rad51 from ssDNA preventing promiscuous strand invasions. Second, it dismantles toxic intermediates that have already formed. Rare survivors in the absence of Srs2 rely on structure-specific endonucleases, Mus81 and Yen1, that resolve toxic joint-molecules. Overall, we uncover a new feature of BIR and propose that tight control of ssDNA accumulated during this process is essential to prevent its channeling into toxic structures threatening cell viability.

Migrating bubble during break-induced replication drives conservative DNA synthesis.

The repair of chromosomal double strand breaks (DSBs) is crucial for the maintenance of genomic integrity. However, the repair of DSBs can also destabilize the genome by causing mutations and chromosomal rearrangements, the driving forces for carcinogenesis and hereditary diseases. Break-induced replication (BIR) is one of the DSB repair pathways that is highly prone to genetic instability. BIR proceeds by invasion of one broken end into a homologous DNA sequence followed by replication that can copy hundreds of kilobases of DNA from a donor molecule all the way through its telomere. The resulting repaired chromosome comes at a great cost to the cell, as BIR promotes mutagenesis, loss of heterozygosity, translocations, and copy number variations, all hallmarks of carcinogenesis. BIR uses most known replication proteins to copy large portions of DNA, similar to S-phase replication. It has therefore been suggested that BIR proceeds by semiconservative replication; however, the model of a bona fide, stable replication fork contradicts the known instabilities associated with BIR such as a 1,000-fold increase in mutation rate compared to normal replication. Here we demonstrate that in budding yeast the mechanism of replication during BIR is significantly different from S-phase replication, as it proceeds via an unusual bubble-like replication fork that results in conservative inheritance of the new genetic material. We provide evidence that this atypical mode of DNA replication, dependent on Pif1 helicase, is responsible for the marked increase in BIR-associated mutations. We propose that the BIR mode of synthesis presents a powerful mechanism that can initiate bursts of genetic instability in eukaryotes, including humans.