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PubMed1976

Mutagen sensitivity of Drosophila melanogaster. III. X-linked loci governing sensitivity to methyl methanesulfonate.

Twenty-eight X-linked, recessive mutations of Drosophila melanogaster conferring enhanced sensitivity to the monofunctional alkylating agent, methyl methanesulfonate, have been recoered and assigned to five complementation groups. These groups can be distinguished on the basis of map location and variations in the pattern of mutagen sensitivity. Allelism of members of one complementation group with the previously described meiotic mutant, mei-41, (Baker and Carpenter, 1972) as well as the frequent appearance of female infertility with mutagen sensitivity suggests associated defects in meiotic chromosome behavior or early embryogenesis. Examination of the mutagen sensitivity of double mutants has led to the formulation of a working model of DNA repair for this organism. Studies of a similar nature (Boyd et al., 1976) have identified five additional X chromosome complementation groups, suggesting that the genome of Drosophila melanogaster may contain many loci involved with mutagen sensitivity. The continued isolation and characterization of conditional mutants of this type promises future insights into the mechanisms of DNA replication, DNA repair and recombination in this complex higher eucaryote.

Animals↗

PubMed2025

Moss BRCA2 lacking the canonical DNA-binding domain promotes homologous recombination and binds to DNA.

BRCA2 is crucial for mediating homology-directed DNA repair (HDR) through its binding to single-stranded DNA (ssDNA) and the recombinases RAD51 and DMC1. Most BRCA2 orthologs have a canonical DNA-binding domain (DBD) with the exception of Drosophila melanogaster. It remains unclear whether such a noncanonical BRCA2 variant without DBD possesses a DNA-binding activity. Here, we identify a new noncanonical BRCA2 in the model plant Physcomitrium patens (PpBRCA2). We establish that PpBRCA2 is essential for genome integrity maintenance, somatic DNA double-strand break (DSB) repair, HDR-mediated gene targeting, and RAD51 foci recruitment at DNA break sites. PpBRCA2 is also critical for DSB repair during meiosis. Interestingly, PpBRCA2 interacts strongly with RAD51 but weakly with DMC1, suggesting a distinct meiotic function compared to other BRCA2 homologs. Despite lacking the canonical DBD, PpBRCA2 binds ssDNA through its disordered N-terminal region and efficiently promotes HDR. Our work highlights that the ssDNA binding capacity of BRCA2 homologs is conserved regardless of the presence of a canonical DBD and provides a deeper understanding of BRCA2's functional diversity across species.

BRCA2 Protein↗

PubMed2025

Gene Editing and Protein Tagging in the Oomycete Phytophthora infestans Using CRISPR-Cas12a.

Molecular genetic tools such as CRISPR-Cas gene editing systems are invaluable for understanding gene and protein function and revealing the details of a pathogen's life and disease cycles. Here we present protocols for genome editing in Phytophthora infestans, an oomycete with global importance as a pathogen of potato and tomato. Using a vector system that expresses variants of Cas12a from Lachnospiraceae bacterium and its guide RNA from a unified transcript, we first present a method for editing genes through the non-homologous end-joining (NHEJ) pathway. We then describe an application of homology-directed repair (HDR), in which Cas12a is used to fuse a protein-coding gene with a fluorescent or epitope tag. Both methods should be adaptable to many oomycetes other than P. infestans.

Gene Editing↗

PubMed2026

Prime assembly with linear DNA donors enables large genomic insertions.

Targeted insertion of large DNA fragments has promising applications for genome engineering and gene therapy1,2. Twin prime-editing guide RNAs have enabled relatively large insertions, but the efficiency remains low for insertions greater than 400 base pairs3-6. Here we describe a prime assembly (PA) approach for the insertion of large DNA donor fragments, of which the ends are designed to overlap with the flaps generated by twin prime editing (twinPE). We used PA to insert one or multiple overlapping DNA fragments, with total insertion sizes ranging from 0.1 kb to 11 kb. An inhibitor of non-homologous end joining enhanced both the efficiency and precision of insertions. PA relies on DNA templates that are easily produced, does not require co-delivery of exogenous DNA-dependent DNA polymerases and proceeds in non-cycling cells, suggesting independence from canonical homology-directed repair pathways. Our study demonstrates that PA can initiate Gibson-like assembly in cells to generate gene insertions without double-stranded DNA breaks, recombinases or homology-directed repair.

Animals↗

PubMed1975

Isolation and characterization of mutants of Haemophilus influenzae deficient in an adenosine 5'-triphosphate-dependent deoxyribonuclease activity.

By a direct assay approach, mutants of Haemophilus influenzae Rd that are deficient in adenosine 5'-triphosphate-dependent deoxyribonuclease activity (add-) were isolated and characterized. A large proportion (50 to 90%) of the cells in cultures of these mutants failed to produce visible colonies when plated. An extensive analysis of the recombination proficiency of these strains revealed that the transformation frequency (transformants per competent cell) in the mutants was similar to that found in the wild type, but that the transformation efficiency (transformants per microgram of irreversibly bound deoxyribonucleic acid [DNA]) was reduced approximately fourfold. Sensitivities of the mutants to gamma rays, ultraviolet radiation, and methyl methane sulfonate were only slightly greater than wild-type levels. The rate of degradation of host DNA after ultraviolet irradiation was significantly reduced in the mutants. It is suggested that the adenosine 5'-triphosphate-dependent deoxyribonuclease in H. influenzae plays a nonessential role in DNA recombination and repair.

Adenosine Triphosphate↗

PubMed1975

Repair of UV-induced DNA damage in recombination-deficient strains of Bacillus subtilis.

To clarify the role of gene products for genetic recombination which might be concerned in excision repair, the repair of DNA lesions induced by ultraviolet (UV) irradiation was examined under non-growing conditions with a variety of recombination deficient (Rec-) mutants of Bacillus subtilis. The extent of repair was estimated by the recovery of transforming activity of DNA extracted from the cells during the post-irradiation incubation period (the assay method was termed as marker-repair experiment). The marker repair seemed to be accomplished by the excision repair as assumed from the effects of inhibitors. Among Rec- mutants tested, a mutant strain UVS80TH (rec-80) exhibited a normal level of marker repair activity, whereas strains GSY1025 (recA1), GSY1028 (recB2) and GSY908 (rec-4) exhibited reduced marker repair activities. These results and the data on UV sensitivity of the mutants indicate that (1) the products of all these Rec genes are related to UV resistance of the cell viability and are factors functioning through mechanisms other than excision repair, (2) the products of recA, recB and rec-4 genes display some roles in maintenance of a level of excision repair activity, and (3) the product of the rec-80 gene does not participate at all in excision repair.

Acriflavine↗

PubMed2025

Distinct roles of the two BRCA2 DNA-binding domains in DNA damage repair and replication fork preservation.

Homologous recombination (HR) removes DNA double-strand breaks (DSBs) and preserves stressed DNA replication forks. Successful HR execution requires the tumor suppressor BRCA2, which harbors distinct DNA-binding domains (DBDs): one that possesses three oligonucleotide/oligosaccharide-binding (OB) folds (OB-DBD) and another residing in the C-terminal recombinase binding domain (CTRB-DBD). Here, we employ multi-faceted approaches to delineate the contributions of these domains toward HR and replication fork maintenance. We show that OB-DBD and CTRB-DBD confer single-strand DNA (ssDNA)- and dsDNA-binding capabilities, respectively, and that BRCA2 variants mutated in either domain are impaired in their ability to load the recombinase RAD51 onto ssDNA pre-occupied by RPA. While the CTRB-DBD mutant is modestly affected by DNA break repair, it exhibits a strong defect in the protection of stressed replication forks. In contrast, the OB-DBD is indispensable for both BRCA2 functions. Our study thus defines the unique contributions of the two BRCA2 DBDs in genome maintenance.

BRCA2 Protein↗

PubMed1976

Isolation of rec- mutants from an F-prime merodiploid strain of Escherichia coli K-12.

This paper describes a method of screening mutagenised populations of an E. coli galA/galB F- prime merodiploid for mutants defective in recombination. The method relies on scoring colonies on Eosin-Methylene Blue agar that have fewer than normal numbers of Gal+ papillae. With a suitable choice of gal- mutations most of the papillae arise by recombination and some of those colonies with less than normal numbers prove to be defective in some aspect of recombination or DNA repair. In addition to strains carrying mutations that can be ascribed to known loci, several novel mutant phenotypes were identified.

Coliphages↗

PubMed2026

GSK3β and Plk1 sequentially phosphorylate ATP-citrate lyase to promote homologous recombination.

Accurate repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is essential for genome stability. Nuclear production of acetyl-coenzyme A (acetyl-CoA) by ATP-citrate lyase (ACLY) promotes HR, yet how ACLY is regulated during the DNA damage response (DDR) remains unclear. Here, we identify a phosphorylation-dependent signaling axis in which glycogen synthase kinase 3β (GSK3β) and Polo-like kinase 1 (Plk1) act sequentially on ACLY to facilitate HR-mediated repair of DSBs induced by ionizing radiation. Following AKT-dependent phosphorylation of ACLY at Ser455, GSK3β phosphorylates ACLY at Thr447, generating a docking site for Plk1, which in turn phosphorylates ACLY at Ser442. This phosphorylation cascade, enhanced by radiation, sustains histone acetylation, supports the accumulation of BRCA1 and RAD51 at DSBs, and confers cellular resistance to poly(ADP-ribose) polymerase (PARP) inhibition. Together, our findings define an AKT-GSK3β-Plk1-ACLY signaling module that links the DDR to nuclear metabolism, revealing a critical mechanism by which kinase signaling facilitates acetyl-CoA-dependent chromatin remodeling to preserve genome integrity.

Protein Serine-Threonine Kinases↗

PubMed2026

Prognostic value and immune landscape implications of using a novel homologous recombination repair pathway signature in prostate cancer: A retrospective cohort study.

ObjectiveAlthough the homologous recombination repair (HRR) pathway plays a critical role in the treatment of prostate cancer, its prognostic value remains incompletely understood. This study aimed to identify HRR pathway-related biomarkers with clinical utility for prognosis prediction and treatment guidance.MethodsWe analyzed genomic data from The Cancer Genome Atlas and Chinese patients with prostate cancer in a retrospective cohort study using a comprehensive multiomics approach to characterize a novel HRR-related prognostic signature and its immune implications.ResultsIn the Chinese cohort, 25.6% of the patients exhibited homologous recombination deficiency scores >42, whereas 27.3% carried ≥1 HRR gene mutation. We established a prognostic HRR signature (homologous recombination deficiency score >32, HRR gene mutations, and Signature 3) associated with poor outcomes. Compared with The Cancer Genome Atlas data, the Chinese cohort demonstrated a higher prevalence of HRR signature. Patients with HRR signatures demonstrated significantly increased genomic instability markers, including segment number, alteration burden, aneuploidy score, and intratumor heterogeneity. The HRR signature was associated with higher neoantigen load but reduced T cell receptor (TCR) evenness. Immunologically, HRR-positive tumors were associated with computationally inferred immune profiles suggestive of reduced immune activity, characterized by depletion of T-helper 17 cell; downregulation of TLR4/PDCD1LG2 expression; and upregulation of ARG1, IFNG, KIR2DL3, and CXCL9. However, these findings are descriptive and require experimental validation.ConclusionOur findings identify a clinically relevant HRR signature that warrants investigation as a potential predictive biomarker for prostate cancer prognosis and treatment response. This biomarker provides new insights for personalized therapy and may help optimize patient outcomes.

Humans↗

PubMed1976

Recombination in Saccharomyces cerevisiae: a DNA repair mutation associated with elevated mitotic gene conversion.

A mutant haploid strain of Saccharomyces cerevisiae has been isolated that is sensitive to the alkylating agent methyl methanesulfonate at a concentration of 0.01% (vol/vol). The strain also shows sensitivities to x-rays and ultra-violet light, which cosegregate with sensitivity to methyl methanesulfonate as a single gene defect. An analysis of the sensitivity to ultraviolet light indicates that the mutation interferes with the excision of pyrimidine dimers. Diploids homozygous for the mutant gene exhibit elevated frequencies of spontaneous mitotic recombination at the ade6 locus. The results indicate that all the events are due to gene conversion. Mitotic recombination was also found to be elevated for three loci other than ade6. Thus, the recombinational effect seems not to be locus specific. Linkage and allelism tests indicate that the mutation is an allele of the known radiation-sensitive gene rad18. The various effects of this new rad18 allele (rad18-3) are discussed in terms of a defect in DNA repair mechanisms.

Alleles↗

PubMed2026

Prevalence of homologous recombination repair genes alterations in metastatic castration-resistant prostate cancer, a multicentric study.

INTRODUCTION: Homologous Recombination Repair (HRR) genes alterations are a resistance mechanism to therapies by taxanes or Androgen Receptor Signalling inhibitors in Metastatic Castration Resistant Prostate Cancer (mCRPC). BRCA-mutated mCRPC patients are eligible to poly adenosine diphosphateribose polymerase inhibitors (PARPi). Therefore, assessing the population-specific prevalence of HRR-related genes alterations is of public healthcare importance. METHODS: This retrospective, non-interventional, multicentric study was conducted across 6 reference French centers in a "real-life" setting. 788 paraffin-embedded mCRPC patient-samples were included and submitted to testing for BRCA1/2 in six different centers; additionally, non-BRCA HRR-related genes were investigated in two different centers. RESULTS: Among the samples, n=602 (76.4%) were contributive for molecular testing. In multivariate analysis by logistic regression and sensitivity analysis, only sample age (P<0.01), sample surface area (P=0.02) and institution (P=0.018) remained statistically significant. BRCA alterations were detected in n=39/602 (6.5%) of contributive samples, with n=35 and n=4 alterations of BRCA2 and BRCA1 respectively. Non-BRCA HRR-related genes alterations were detected in n=12/157 (7.6%) of contributive samples, with alterations of mainly ATM (n=6, 3.8%), CDK12 (n=4, 2.5%) and CHEK2 (n=2, 1.3%). DISCUSSION: In this study, testing contributivity was similar or higher that of other studies in the literature, and observed mutations prevalences were similar to that of other screenings of western populations. Harmonising per-centres protocols and enhancing molecular testing contributivity with the screening of circulating DNA samples and expanding its range by including non-BRCA HRR-related genes in all reference centres will enable more patients to be accurately treated by targeted therapies. LEVEL OF EVIDENCE: 3 (grade C).

Male↗

PubMed2015

NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability.

NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.

Cell Line↗

PubMed2026

Impact of homologous recombination repair gene mutations on survival in metastatic prostate cancer: A real-world analysis from an observational database.

The prognostic significance of homologous recombination repair gene (HRRg) mutations across the different metastatic prostate cancer stages remains unclear. This retrospective real-world study analyzed 162 metastatic castration-sensitive (mCSPC) and 126 castration-resistant (mCRPC) patients from the ProGène database, stratified by HRRg mutational status. Mutation prevalence was similar in both groups (16.0% in mCSPC vs. 13.5% in mCRPC). HRR-positive mCSPC patients had significantly shorter median overall survival (OS) (24.0months; 95% confidence interval [CI]: 16.0-41.0) compared to HRR-negative patients (45.0months; 95% CI: 34.0-69.0; P=0.04). Notably, BRCA2-mutated patients exhibited a reduced median OS of 24.0months (95% CI: 9.0-40.0; P=0.036) and a faster progression-free survival compared to HRR-negative patients (median PFS=8.0months; 95% CI: 0.0-14.0 vs. 17.0months; 95% CI: 12.0-20.0; P=0.006). These findings suggest that HRRg mutations - especially BRCA2 - are associated with worse prognosis in mCSPC, supporting the value of early genomic screening to guide personalized treatment strategies.

Humans↗

PubMed2025

SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.

Generating nonessential gene knockouts using CRISPR/Cas9 technology is becoming increasingly common in biological research. In a typical workflow, the Cas9 endonuclease is used to induce a DNA double-strand break that relies on nonhomologous end-joining (NHEJ) to introduce a premature termination codon (PTC) in the target gene. The goal is to isolate clones in which the gene produces PTC-containing mRNA transcripts that are degraded via nonsense-mediated mRNA decay (NMD) to cause loss of gene function. Unfortunately, this approach is laborious, and not all PTCs trigger NMD. More importantly, mounting evidence suggest that PTC mutations can also result in a transcriptional adaptation response that can mask the effects of a PTC-mediated gene knockout. In this chapter, we present a PTC-free gene knockout strategy that implements homology-directed DNA repair (HDR) with selectable markers to substantially reduce the complexity of the screening and validation of genome edits in cells containing more than one gene copy as in the case of the commonly used hypotriploid HEK293 cell line. We describe how to obtain a complete knockout of the Ligase IV protein (LIG4) and provide considerations for the application of this SelectRepair Knockout method to other genes.

Humans↗

PubMed2026

Transgenerational chromosome repair in the asexual bdelloid rotifer Adineta vaga.

Homologous recombination is an essential DNA repair mechanism that also promotes chromosome pairing and ensures allele segregation during meiosis in sexual organisms. Here, we explore the dual function of homologous recombination in the bdelloid rotifer Adineta vaga, an asexual species known for its remarkable resilience to extreme genotoxic stresses. Genomic analyses reveal that A. vaga uses meiotic recombination to promote spontaneous crossovers and gene conversion during oogenesis and to repair the genome in response to DNA damage. The data also support a model of transgenerational DNA repair, termed break-induced homologous extension repair (BIHER), in which broken chromosomes are progressively restored over multiple generations. Our findings suggest that meiotic BIHER, coupled with the holocentric structure of chromosomes, may represent a key adaptation of life in extreme environments.

Animals↗

PubMed2026

Cohesin promotes genomic stability by suppressing unequal sister chromatid exchange.

The protein complex cohesin plays critical roles in genomic stability by tethering together sister chromatids at their pericentric regions and along their arms from S phase until anaphase. Cohesin-mediated pericentric cohesion prevents aneuploidy by ensuring bipolar attachment of sister kinetochores. Arm cohesion prevents loss of heterozygosity by biasing DNA repair via recombination between sister chromatids rather than between homologs. Here, we investigate in yeast whether cohesin also enhances genomic stability by suppressing unequal sister chromatid exchange (USCE) between repetitive sequences. In wild-type cells, the USCE rate between repeats 4kb apart (proximal) was 15X higher than repeats 68kb apart (distal). The USCE between distal repeats but not proximal repeats increased 4 to 7-fold in mutants with altered cohesin subunits or auxiliary factors. The level of increased distal USCE corresponded with reduced arm cohesion, reduced density of cohesion arm sites, and higher sister loci mobility. Our results suggest that high density of arm cohesion sites confines repair of DNA damage to local sequences. When the density of cohesion sites decreases, sister chromatid sequences are less confined, thereby enhancing distal repeat interactions and USCE. Another set of mutations disrupted both DNA replication and cohesin loading at the replication fork during S phase. Remarkably, distal USCE in these mutants increased approximately 100-fold and was 6-fold more likely than proximal USCE. This preferential hyperdistal USCE can be explained by an aberrant sister-chromatid structure that is normally prevented by proper coupling of cohesin function and replication.

Journal Article↗

PubMed2019

Homologous recombination defects and how they affect replication fork maintenance.

Homologous recombination (HR) repairs DNA double strand breaks (DSBs) and stabilizes replication forks (RFs). RAD51 is the recombinase for the HR pathway. To preserve genomic integrity, RAD51 forms a filament on the 3' end of a DSB and on a single-stranded DNA (ssDNA) gap. But unregulated HR results in undesirable chromosomal rearrangements. This review describes the multiple mechanisms that regulate HR with a focus on those mechanisms that promote and contain RAD51 filaments to limit chromosomal rearrangements. If any of these pathways break down and HR becomes unregulated then disease, primarily cancer, can result.

RAD51 filaments↗