EXO1 protects BRCA1-deficient cells against toxic DNA lesions.

Inactivating mutations in the BRCA1 and BRCA2 genes impair DNA double-strand break (DSB) repair by homologous recombination (HR), leading to chromosomal instability and cancer. Importantly, BRCA1/2 deficiency also causes therapeutically targetable vulnerabilities. Here, we identify the dependency on the end resection factor EXO1 as a key vulnerability of BRCA1-deficient cells. EXO1 deficiency generates poly(ADP-ribose)-decorated DNA lesions during S phase that associate with unresolved DSBs and genomic instability in BRCA1-deficient but not in wild-type or BRCA2-deficient cells. Our data indicate that BRCA1/EXO1 double-deficient cells accumulate DSBs due to impaired repair by single-strand annealing (SSA) on top of their HR defect. In contrast, BRCA2-deficient cells retain SSA activity in the absence of EXO1 and hence tolerate EXO1 loss. Consistent with a dependency on EXO1-mediated SSA, we find that BRCA1-mutated tumors show elevated EXO1 expression and increased SSA-associated genomic scars compared with BRCA1-proficient tumors. Overall, our findings uncover EXO1 as a promising therapeutic target for BRCA1-deficient tumors.

FANCA Promotes DNA Double-Strand Break Repair by Catalyzing Single-Strand Annealing and Strand Exchange.

FANCA is a component of the Fanconi anemia (FA) core complex that activates DNA interstrand crosslink repair by monoubiquitination of FANCD2. Here, we report that purified FANCA protein catalyzes bidirectional single-strand annealing (SA) and strand exchange (SE) at a level comparable to RAD52, while a disease-causing FANCA mutant, F1263Δ, is defective in both activities. FANCG, which directly interacts with FANCA, dramatically stimulates its SA and SE activities. Alternatively, FANCB, which does not directly interact with FANCA, does not stimulate this activity. Importantly, five other patient-derived FANCA mutants also exhibit deficient SA and SE, suggesting that the biochemical activities of FANCA are relevant to the etiology of FA. A cell-based DNA double-strand break (DSB) repair assay demonstrates that FANCA plays a direct role in the single-strand annealing sub-pathway (SSA) of DSB repair by catalyzing SA, and this role is independent of the canonical FA pathway and RAD52.

A glimpse into the hidden world of the flexible C-terminal protein binding domains of human RAD52.

Human RAD52 protein binds DNA and is involved in genomic stability maintenance and several forms of DNA repair, including homologous recombination and single-strand annealing. Despite its importance, there are very few structural details about the variability of the RAD52 ring size and the RAD52 C-terminal protein-protein interaction domains. Even recent attempts to employ cryogenic electron microscopy (cryoEM) methods on full-length yeast and human RAD52 do not reveal interpretable structures for the C-terminal half that contains the replication protein A (RPA) and RAD51 binding domains. In this study, we employed the monodisperse purification of two RAD52 deletion constructs and small angle X-ray scattering (SAXS) to construct a structural model that includes RAD52's RPA binding domain. This model is of interest to DNA repair specialists as well as for drug development against HR-deficient cancers.

Natural transformation-specific DprA coordinate DNA double-strand break repair pathways in heavily irradiated D. radiodurans.

Deinococcus radiodurans exhibits remarkable survival under extreme conditions, including ionizing radiation, desiccation, and various DNA-damaging agents. It employs unique repair mechanisms, such as single-strand annealing (SSA) and extended synthesis-dependent strand annealing (ESDSA), to efficiently restore damaged genome. In this study, we investigate the role of the natural transformation-specific protein DprA in DNA repair pathways following acute gamma radiation exposure. Our findings demonstrate that the absence of DprA leads to rapid repair of gamma radiation-induced DNA double-strand breaks primarily occur through SSA repair pathway. Additionally, our findings suggest that the DprA protein may hinder both the SSA and ESDSA repair pathways, albeit in distinct manners. Overall, our results highlight the crucial function of DprA in the selection between SSA and ESDSA pathways for DNA repair in heavily irradiated D. radiodurans.IMPORTANCEDeinococcus radiodurans exhibits an extraordinary ability to endure and thrive in extreme environments, including exposure to radiation, desiccation, and damaging chemicals, as well as intense UV radiation. The bacterium has evolved highly efficient repair mechanisms capable of rapidly mending hundreds of DNA fragments in its genome. Our research indicates that natural transformation (NT)-specific dprA genes play a pivotal role in regulating DNA repair in response to radiation. Remarkably, we found that DprA is instrumental in selecting DNA double-strand break repair pathways, a novel function that has not been reported before. This unique regulatory mechanism highlights the indispensable role of DprA beyond its native function in NT and underscores its ubiquitous presence across various bacterial species, regardless of their NT proficiency. These findings shed new light on the resilience and adaptability of Deinococcus radiodurans, opening avenues for further exploration into its exceptional survival strategies.

Break-induced replication plays a prominent role in long-range repeat-mediated deletion.

Repetitive DNA sequences are often associated with chromosomal rearrangements in cancers. Conventionally, single-strand annealing (SSA) is thought to mediate homology-directed repair of double-strand breaks (DSBs) between two repeats, causing repeat-mediated deletion (RMD). In this report, we demonstrate that break-induced replication (BIR) is used predominantly over SSA in mammalian cells for mediating RMD, especially when repeats are far apart. We show that SSA becomes inefficient in mammalian cells when the distance between the DSBs and the repeats is increased to the 1-2 kb range, while BIR-mediated RMD (BIR/RMD) can act over a long distance (e.g., ~ 100-200 kb) when the DSB is close to one repeat. Importantly, oncogene expression potentiates BIR/RMD but not SSA, and BIR/RMD is used more frequently at single-ended DSBs formed at collapsed replication forks than at double-ended DSBs. In contrast to short-range SSA, H2AX is required for long-range BIR/RMD, and sequence divergence strongly suppresses BIR/RMD in a manner partially dependent on MSH2. Our finding that BIR/RMD has a more important role than SSA in mammalian cells has a significant impact on the understanding of repeat-mediated rearrangements associated with oncogenesis.

A Boolean network model of the double-strand break repair pathway choice.

Double strand break (DSB) repair is critical to maintaining the integrity of the genome. DSB repair deficiency underlies multiple pathologies, including cancer, chromosome instability syndromes, and, potentially, neurodevelopmental defects. DSB repair is mainly handled by two pathways: highly accurate homologous recombination (HR), which requires a sister chromatid for template-based repair, limited to S/G2 phases of the cell cycle, and canonical non-homologous end joining (c-NHEJ), available throughout the cell cycle in which minimum homology is sufficient for highly efficient yet error-prone repair. Some circumstances, such as cancer, require alternative highly mutagenic DSB repair pathways like microhomology-mediated end-joining (MMEJ) and single-strand annealing (SSA), which are triggered to attend to DNA damage. These non-canonical repair alternatives are emerging as prominent drivers of resistance in drug-based tumor therapies. Multiple DSB repair options require tight inter-pathway regulation to prevent unscheduled activities. In addition to this complexity, epigenetic modifications of the histones surrounding the DSB region are emerging as critical regulators of the DSB repair pathway choice. Modeling approaches to understanding DSBs repair pathway choice are advantageous to perform simulations and generate predictions on previously uncharacterized aspects of DSBs response. In this work, we present a Boolean network model of the DSB repair pathway choice that incorporates the knowledge, into a dynamic system, of the inter-pathways regulation involved in DSB repair, i.e., HR, c-NHEJ, SSA, and MMEJ. Our model recapitulates the well-characterized HR activity observed in wild-type cells in response to DSBs. It also recovers clinically relevant behaviors of BRCA1/FANCS mutants, and their corresponding drug resistance mechanisms ascribed to DNA repair gain-of-function pathogenic variants. Since epigenetic modifiers are dynamic and possible druggable targets, we incorporated them into our model to better characterize their involvement in DSB repair. Our model predicted that loss of the TIP60 complex and its corresponding histone acetylation activity leads to activation of SSA in response to DSBs. Our experimental validation showed that TIP60 effectively prevents activation of RAD52, a key SSA executor, and confirms the suitable use of Boolean network modeling for understanding DNA DSB repair.

The pathway of recombining short homologous ends in Escherichia coli revealed by the genetic study.

The recombination of short homologous ends in Escherichia coli has been known for 30 years, and it is often used for both site-directed mutagenesis and in vivo cloning. For cloning, a plasmid and target DNA fragments were converted into linear DNA fragments with short homologous ends, which are joined via recombination inside E. coli after transformation. Here this mechanism of joining homologous ends in E. coli was determined by a linearized plasmid with short homologous ends. Two 3'-5' exonucleases ExoIII and ExoX with nonprocessive activity digested linear dsDNA to generate 5' single-strand overhangs, which annealed with each other. The polymerase activity of DNA polymerase I (Pol I) was exclusively employed to fill in the gaps. The strand displacement activity and the 5'-3' exonuclease activity of Pol I were also required, likely to generate 5' phosphate termini for subsequent ligation. Ligase A (LigA) joined the nicks to finish the process. The model involving 5' single-stranded overhangs is different from established recombination pathways that all generate 3' single-stranded overhangs. This recombination is likely common in bacteria since the involved enzymes are ubiquitous.

Frequency of DNA end joining in trans is not determined by the predamage spatial proximity of double-strand breaks in yeast.

DNA double-strand breaks (DSBs) are serious genomic insults that can lead to chromosomal rearrangements if repaired incorrectly. To gain insight into the nuclear mechanisms contributing to these rearrangements, we developed an assay in yeast to measure cis (same site) vs. trans (different site) repair for the majority process of precise nonhomologous end joining (NHEJ). In the assay, the HO endonuclease gene is placed between two HO cut sites such that HO expression is self-terminated upon induction. We further placed an additional cut site in various genomic loci such that NHEJ in trans led to expression of a LEU2 reporter gene. Consistent with prior reports, cis NHEJ was more efficient than trans NHEJ. However, unlike homologous recombination, where spatial distance between a single DSB and donor locus was previously shown to correlate with repair efficiency, trans NHEJ frequency remained essentially constant regardless of the position of the two DSB loci, even when they were on the same chromosome or when two trans repair events were put in competition. Repair of similar DSBs via single-strand annealing of short terminal direct repeats showed substantially higher repair efficiency and trans repair frequency, but still without a strong correlation of trans repair to genomic position. Our results support a model in which yeast cells mobilize, and perhaps compartmentalize, multiple DSBs in a manner that no longer reflects the predamage position of two broken loci.

Precision genome editing in plants via gene targeting and subsequent break-induced single-strand annealing.

Genome editing via artificial nucleases such as CRISPR/Cas9 has become popular in plants now. However, small insertions or deletions are major mutations and nucleotide substitutions rarely occur when DNA cleavage is induced. To induce nucleotide substitutions, a base editor utilizing dead or nickase-type Cas9 fused with deaminase have been developed. However, the direction and position of practical substitution are still limited. In this context, homologous recombination (HR)-mediated gene targeting (GT) has advantages because any mutations existing on the donor DNA are copied and passed onto the endogenous DNA. As HR-mediated GT is extremely rare in higher plants, positive-negative selection has been used to isolate cells in which GT has occurred. After successful selection, positive selection marker is no longer needed and should ideally be eliminated. In a previous study, we reported a seamless piggyBac-transposon-mediated marker elimination system. Precision marker elimination efficiency in this system is very high. The piggyBac transposon integrates into the host genome at TTAA elements and excises without leaving a footprint at the excised site, so a TTAA sequence is necessary at the location of a positive selection marker. To compensate for this limitation, we have developed a novel marker elimination system using an I-SceI break and subsequent single-strand annealing (SSA)-mediated DNA repair system.

RNF169 limits 53BP1 deposition at DSBs to stimulate single-strand annealing repair.

Unrestrained 53BP1 activity at DNA double-strand breaks (DSBs) hampers DNA end resection and upsets DSB repair pathway choice. RNF169 acts as a molecular rheostat to limit 53BP1 deposition at DSBs, but how this fine balance translates to DSB repair control remains undefined. In striking contrast to 53BP1, ChIP analyses of AsiSI-induced DSBs unveiled that RNF169 exhibits robust accumulation at DNA end-proximal regions and preferentially targets resected, RPA-bound DSBs. Accordingly, we found that RNF169 promotes CtIP-dependent DSB resection and favors homology-mediated DSB repair, and further showed that RNF169 dose-dependently stimulates single-strand annealing repair, in part, by alleviating the 53BP1-imposed barrier to DSB end resection. Our results highlight the interplay of RNF169 with 53BP1 in fine-tuning choice of DSB repair pathways.

Mutational Analysis of Redß Single Strand Annealing Protein: Roles of the 14 Lysine Residues in DNA Binding and Recombination In Vivo.

Redß is a 261 amino acid protein from bacteriophage λ that promotes a single-strand annealing (SSA) reaction for repair of double-stranded DNA (dsDNA) breaks. While there is currently no high-resolution structure available for Redß, models of its DNA binding domain (residues 1-188) have been proposed based on homology with human Rad52, and a crystal structure of its C-terminal domain (CTD, residues 193-261), which binds to λ exonuclease and E. coli single-stranded DNA binding protein (SSB), has been determined. To evaluate these models, the 14 lysine residues of Redß were mutated to alanine, and the variants tested for recombination in vivo and DNA binding and annealing in vitro. Most of the lysines within the DNA binding domain, including K36, K61, K111, K132, K148, K154, and K172, were found to be critical for DNA binding in vitro and recombination in vivo. By contrast, none of the lysines within the CTD, including K214, K245, K251, K253, and K258 were required for DNA binding in vitro, but two, K214 and K253, were critical for recombination in vivo, likely due to their involvement in binding to SSB. K61 was identified as a residue that is critical for DNA annealing, but not for initial ssDNA binding, suggesting a role in binding to the second strand of DNA incorporated into the complex. The K148A variant, which has previously been shown to be defective in oligomer formation, had the lowest affinity for ssDNA, and was the only variant that was completely non-cooperative, suggesting that ssDNA binding is coupled to oligomerization.

Single-Strand Annealing in Cancer.

DNA double-strand breaks (DSBs) are among the most serious forms of DNA damage. In humans, DSBs are repaired mainly by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). Single-strand annealing (SSA), another DSB repair system, uses homologous repeats flanking a DSB to join DNA ends and is error-prone, as it removes DNA fragments between repeats along with one repeat. Many DNA deletions observed in cancer cells display homology at breakpoint junctions, suggesting the involvement of SSA. When multiple DSBs occur in different chromosomes, SSA may result in chromosomal translocations, essential in the pathogenesis of many cancers. Inhibition of RAD52 (RAD52 Homolog, DNA Repair Protein), the master regulator of SSA, results in decreased proliferation of BRCA1/2 (BRCA1/2 DNA Repair Associated)-deficient cells, occurring in many hereditary breast and ovarian cancer cases. Therefore, RAD52 may be targeted in synthetic lethality in cancer. SSA may modulate the response to platinum-based anticancer drugs and radiation. SSA may increase the efficacy of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated 9) genome editing and reduce its off-target effect. Several basic problems associated with SSA, including its evolutionary role, interplay with HRR and NHEJ and should be addressed to better understand its role in cancer pathogenesis and therapy.

Disruption of Chromatin Dynamics by Hypotonic Stress Suppresses HR and Shifts DSB Processing to Error-Prone SSA.

The processing of DNA double-strand breaks (DSBs) depends on the dynamic characteristics of chromatin. To investigate how abrupt changes in chromatin compaction alter these dynamics and affect DSB processing and repair, we exposed irradiated cells to hypotonic stress (HypoS). Densitometric and chromosome-length analyses show that HypoS transiently decompacts chromatin without inducing histone modifications known from regulated local chromatin decondensation, or changes in Micrococcal Nuclease (MNase) sensitivity. HypoS leaves undisturbed initial stages of DNA-damage-response (DDR), such as radiation-induced ATM activation and H2AX-phosphorylation. However, detection of ATM-pS1981, γ-H2AX and 53BP1 foci is reduced in a protein, cell cycle phase and cell line dependent manner; likely secondary to chromatin decompaction that disrupts the focal organization of DDR proteins. While HypoS only exerts small effects on classical nonhomologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ), it markedly suppresses homologous recombination (HR) without affecting DNA end-resection at DSBs, and clearly enhances single-strand annealing (SSA). These shifts in pathway engagement are accompanied by decreases in HR-dependent chromatid-break repair in the G2-phase, and by increases in alt-EJ and SSA-dependent chromosomal translocations. Consequently, HypoS sensitizes cells to ionizing radiation (IR)-induced killing. We conclude that HypoS-induced global chromatin decompaction compromises regulated chromatin dynamics and genomic stability by suppressing DSB-processing by HR, and allowing error-prone processing by alt-EJ and SSA.

Protein-Assisted Room-Temperature Assembly of Rigid, Immobile Holliday Junctions and Hierarchical DNA Nanostructures.

Immobile Holliday junctions represent not only the most fundamental building block of structural DNA nanotechnology but are also of tremendous importance for the in vitro investigation of genetic recombination and epigenetics. Here, we present a detailed study on the room-temperature assembly of immobile Holliday junctions with the help of the single-strand annealing protein Redß. Individual DNA single strands are initially coated with protein monomers and subsequently hybridized to form a rigid blunt-ended four-arm junction. We investigate the efficiency of this approach for different DNA/protein ratios, as well as for different DNA sequence lengths. Furthermore, we also evaluate the potential of Redß to anneal sticky-end modified Holliday junctions into hierarchical assemblies. We demonstrate the Redß-mediated annealing of Holliday junction dimers, multimers, and extended networks several microns in size. While these hybrid DNA-protein nanostructures may find applications in the crystallization of DNA-protein complexes, our work shows the great potential of Redß to aid in the synthesis of functional DNA nanostructures under mild reaction conditions.

Repair of DNA double-strand breaks by mammalian alternative end-joining pathways.

Alternative end-joining (a-EJ) pathways, which repair DNA double-strand breaks (DSBs), are initiated by end resection that generates 3' single strands. This reaction is shared, at least in part, with homologous recombination but distinguishes a-EJ from the major nonhomologous end-joining pathway. Although the a-EJ pathways make only a minor and poorly understood contribution to DSB repair in nonmalignant cells, there is growing interest in these pathways, as they generate genomic rearrangements that are hallmarks of cancer cells. Here, we review and discuss the current understanding of the mechanisms and regulation of a-EJ pathways, the role of a-EJ in human disease, and the potential utility of a-EJ as a therapeutic target in cancer.

Regulation of Single-Strand Annealing and its Role in Genome Maintenance.

Single-strand annealing (SSA) is a DNA double-strand break (DSB) repair pathway that uses homologous repeats to bridge DSB ends. SSA involving repeats that flank a single DSB causes a deletion rearrangement between the repeats, and hence is relatively mutagenic. Nevertheless, this pathway is conserved, in that SSA events have been found in several organisms. In this review, we describe the mechanism of SSA and its regulation, including the cellular conditions that may favor SSA versus other DSB repair events. We will also evaluate the potential contribution of SSA to cancer-associated genome rearrangements, and to DSB-induced gene targeting.

Trinucleotide repeat instability during double-strand break repair: from mechanisms to gene therapy.

Trinucleotide repeats are a particular class of microsatellites whose large expansions are responsible for at least two dozen human neurological and developmental disorders. Slippage of the two complementary DNA strands during replication, homologous recombination or DNA repair is generally accepted as a mechanism leading to repeat length changes, creating expansions and contractions of the repeat tract. The present review focuses on recent developments on double-strand break repair involving trinucleotide repeat tracts. Experimental evidences in model organisms show that gene conversion and break-induced replication may lead to large repeat tract expansions, while frequent contractions occur either by single-strand annealing between repeat ends or by gene conversion, triggering near-complete contraction of the repeat tract. In the second part of this review, different therapeutic approaches using highly specific single- or double-strand endonucleases targeted to trinucleotide repeat loci are compared. Relative efficacies and specificities of these nucleases will be discussed, as well as their potential strengths and weaknesses for possible future gene therapy of these dramatic disorders.

Recombination promoted by DNA viruses: phage λ to herpes simplex virus.

The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies.

Pathways and assays for DNA double-strand break repair by homologous recombination.

Double strand breaks (DSBs) are the most detrimental type of DNA damage that must be repaired to ensure genome integrity and cell survival. Unrepaired or improperly repaired DSBs can potentially cause tumorigenesis or cell death. DSBs are primarily repaired by non-homologous end joining or homologous recombination (HR). The HR pathway is initiated by processing of the 5'-end of DSBs to generate 3'-end single-strand DNA (ssDNA). Furthermore, the intermediate is channeled to one of the HR sub-pathways, including: (i) double Holliday junction (dHJ) pathway, (ii) synthesis-dependent strand annealing (SDSA), (iii) break-induced replication (BIR), and (iv) single-strand annealing (SSA). In the dHJ sub-pathway, the 3'-ssDNA coated with Rad51 recombinase performs homology search and strand invasion, forming a displacement loop (D-loop). Capture of the second end by the D-loop generates a dHJ intermediate that is subsequently dissolved by DNA helicase or resolved by nucleases, producing non-crossover or crossover products. In SDSA, the newly synthesized strand is displaced from the D-loop and anneals to the end on the other side of the DSBs, producing non-crossovers. In contrast, BIR repairs one-end DSBs by copying the sequence up to the end of the template chromosome, resulting in translocation or loss of heterozygosity. SSA takes place when resection reveals flanking homologous repeats that can anneal, leading to deletion of the intervening sequences. A variety of reporter assays have been developed to monitor distinct HR sub-pathways in both Saccharomyces cerevisiae and mammals. Here, we summarize the principles and representative assays for different HR sub-pathways with an emphasis on the studies in the budding yeast.

Mechanisms of germ line genome instability.

During meiosis, numerous DNA double-strand breaks (DSBs) are formed as part of the normal developmental program. This seemingly destructive behavior is necessary for successful meiosis, since repair of the DSBs through homologous recombination (HR) helps to produce physical links between the homologous chromosomes essential for correct chromosome segregation later in meiosis. However, DSB formation at such a massive scale also introduces opportunities to generate gross chromosomal rearrangements. In this review, we explore ways in which meiotic DSBs can result in such genomic alterations.