Homologous recombination involves the formation of branched DNA molecules that may interfere with chromosome segregation. To resolve these persistent joint molecules, cells rely on the activation of structure-selective endonucleases (SSEs) during the late stages of the cell cycle. However, the premature activation of SSEs compromises genome integrity, due to untimely processing of replication and/or recombination intermediates. Here, we used a biochemical approach to show that the budding yeast SSEs Mus81 and Yen1 possess the ability to cleave the central recombination intermediate known as the displacement loop or D-loop. Moreover, we demonstrate that, consistently with previous genetic data, the simultaneous action of Mus81 and Yen1, followed by ligation, is sufficient to recreate the formation of a half-crossover precursor in vitro. Our results provide not only mechanistic explanation for the formation of a half-crossover, but also highlight the critical importance for precise regulation of these SSEs to prevent chromosomal rearrangements.

• MeSH
• Crossing Over, Genetic * MeSH
• DNA-Binding Proteins * metabolism   genetics   MeSH
• Endonucleases * metabolism   genetics   MeSH
• Homologous Recombination MeSH
• Holliday Junction Resolvases metabolism   genetics   MeSH
• Saccharomyces cerevisiae Proteins * metabolism   genetics   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA-Binding Proteins * MeSH
• Endonucleases * MeSH
• MUS81 protein, S cerevisiae MeSH Browser
• Holliday Junction Resolvases MeSH
• Saccharomyces cerevisiae Proteins * MeSH
• Yen1 protein, S cerevisiae MeSH Browser

The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that 'nucleotide proofreading' activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.

• MeSH
• Adenosine Diphosphate pharmacology   MeSH
• Adenosine Triphosphate pharmacology   MeSH
• Caenorhabditis elegans metabolism   MeSH
• Species Specificity MeSH
• Fluorescence MeSH
• Interferometry MeSH
• DNA, Single-Stranded metabolism   MeSH
• Nucleotides metabolism   MeSH
• Caenorhabditis elegans Proteins metabolism   MeSH
• Rad51 Recombinase chemistry   metabolism   MeSH
• Sequence Homology, Amino Acid * MeSH
• Protein Stability drug effects   MeSH
• Protein Binding drug effects   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Diphosphate MeSH
• Adenosine Triphosphate MeSH
• DNA, Single-Stranded MeSH
• Nucleotides MeSH
• Caenorhabditis elegans Proteins MeSH
• Rad51 Recombinase MeSH

The evolutionarily conserved Swi5-Sfr1 complex plays an important role in homologous recombination, a process crucial for the maintenance of genomic integrity. Here, we purified Schizosaccharomyces pombe Swi5-Sfr1 complex from meiotic cells and analyzed it by mass spectrometry. Our analysis revealed new phosphorylation sites on Swi5 and Sfr1. We found that mutations that prevent phosphorylation of Swi5 and Sfr1 do not impair their function but swi5 and sfr1 mutants encoding phosphomimetic aspartate at the identified phosphorylation sites are only partially functional. We concluded that during meiosis, Swi5 associates with Sfr1 and both Swi5 and Sfr1 proteins are phosphorylated. However, the functional relevance of Swi5 and Sfr1 phosphorylation remains to be determined.

• Keywords
• DNA repair, Schizosaccharomyces pombe, Sfr1, Swi5, meiosis, phosphorylation, recombination,
• MeSH
• Phosphorylation MeSH
• Homologous Recombination * MeSH
• Meiosis MeSH
• DNA Repair * MeSH
• DNA Damage * MeSH
• Schizosaccharomyces pombe Proteins genetics   metabolism   MeSH
• Schizosaccharomyces genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Schizosaccharomyces pombe Proteins MeSH
• Sfr1 protein, S pombe MeSH Browser
• Swi5 protein, S pombe MeSH Browser

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51-ssDNA filaments. RECQ5 interacts with RAD51 through protein-protein contacts, and disruption of this interface through a RECQ5-F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51-K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51-I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Point Mutation MeSH
• RecQ Helicases genetics   metabolism   ultrastructure   MeSH
• Homologous Recombination * MeSH
• Hydrolysis MeSH
• DNA, Single-Stranded metabolism   ultrastructure   MeSH
• Kinetics MeSH
• Humans MeSH
• Microscopy, Atomic Force MeSH
• Mutation, Missense MeSH
• Molecular Motor Proteins metabolism   ultrastructure   MeSH
• Recombinant Fusion Proteins metabolism   MeSH
• Recombinant Proteins metabolism   MeSH
• Rad51 Recombinase genetics   metabolism   MeSH
• Replication Protein A metabolism   MeSH
• Substrate Specificity MeSH
• Single Molecule Imaging * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• RecQ Helicases MeSH
• DNA, Single-Stranded MeSH
• Molecular Motor Proteins MeSH
• RAD51 protein, human MeSH Browser
• RECQL5 protein, human MeSH Browser
• Recombinant Fusion Proteins MeSH
• Recombinant Proteins MeSH
• Rad51 Recombinase MeSH
• Replication Protein A MeSH
• RPA1 protein, human MeSH Browser

DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). RAD18/UBC13/three prime repair exonuclease 2 (TREX2)-mediated proliferating cell nuclear antigen (PCNA) ubiquitination is central to DDT, an error-prone lesion bypass pathway. RAD51 is the recombinase for HR. The RAD51 K133A mutation increased spontaneous mutations and stress-induced RF stalls and nascent strand degradation. Here, we report in RAD51K133A cells that this phenotype is reduced by expressing a TREX2 H188A mutation that deletes its exonuclease activity. In RAD51K133A cells, knocking out RAD18 or overexpressing PCNA reduces spontaneous mutations, while expressing ubiquitination-incompetent PCNAK164R increases mutations, indicating DDT as causal. Deleting TREX2 in cells deficient for the RF maintenance proteins poly(ADP-ribose) polymerase 1 (PARP1) or FANCB increased nascent strand degradation that was rescued by TREX2H188A, implying that TREX2 prohibits degradation independent of catalytic activity. A possible explanation for this occurrence is that TREX2H188A associates with UBC13 and ubiquitinates PCNA, suggesting a dual role for TREX2 in RF maintenance.

• Keywords
• DNA damage tolerance, double-strand break repair, genomic instability, homologous recombination, replication fork maintenance,
• MeSH
• Exodeoxyribonucleases genetics   metabolism   MeSH
• Phosphoproteins genetics   metabolism   MeSH
• Humans MeSH
• Mutation * MeSH
• Mice MeSH
• Rad51 Recombinase biosynthesis   genetics   metabolism   MeSH
• DNA Replication * MeSH
• Transfection MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Exodeoxyribonucleases MeSH
• Phosphoproteins MeSH
• RAD51 protein, human MeSH Browser
• Rad51 Recombinase MeSH
• TREX2 protein, human MeSH Browser

RECQ5 belongs to the RecQ family of DNA helicases. It is conserved from Drosophila to humans and its deficiency results in genomic instability and cancer susceptibility in mice. Human RECQ5 is known for its ability to regulate homologous recombination by disrupting RAD51 nucleoprotein filaments. It also binds to RNA polymerase II (RNAPII) and negatively regulates transcript elongation by RNAPII. Here, we summarize recent studies implicating RECQ5 in the prevention and resolution of transcription-replication conflicts, a major intrinsic source of genomic instability during cancer development.

• Keywords
• DNA repair, R-loops, RECQ5, genomic instability, replication stress, transcription-replication conflicts,
• MeSH
• DNA genetics   metabolism   MeSH
• Transcription, Genetic genetics   MeSH
• RecQ Helicases genetics   metabolism   physiology   MeSH
• Humans MeSH
• Genomic Instability MeSH
• DNA Replication MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• DNA MeSH
• RecQ Helicases MeSH

Formation of RAD51 filaments on single-stranded DNA is an essential event during homologous recombination, which is required for homology search, strand exchange and protection of replication forks. Formation of nucleoprotein filaments (NF) is required for development and genomic stability, and its failure is associated with developmental abnormalities and tumorigenesis. Here we describe the structure of the human RAD51 NFs and of its Walker box mutants using electron microscopy. Wild-type RAD51 filaments adopt an 'open' conformation when compared to a 'closed' structure formed by mutants, reflecting alterations in helical pitch. The kinetics of formation/disassembly of RAD51 filaments show rapid and high ssDNA coverage via low cooperativity binding of RAD51 units along the DNA. Subsequently, a series of isomerization or dissociation events mediated by nucleotide binding state creates intrinsically dynamic RAD51 NFs. Our findings highlight important a mechanistic divergence among recombinases from different organisms, in line with the diversity of biological mechanisms of HR initiation and quality control. These data reveal unexpected intrinsic dynamic properties of the RAD51 filament during assembly/disassembly, which may be important for the proper control of homologous recombination.

• MeSH
• Adenine Nucleotides metabolism   MeSH
• Adenosine Triphosphate metabolism   MeSH
• Biological Evolution MeSH
• Cryoelectron Microscopy MeSH
• DNA, Single-Stranded metabolism   MeSH
• Kinetics MeSH
• Humans MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Rad51 Recombinase genetics   metabolism   ultrastructure   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenine Nucleotides MeSH
• Adenosine Triphosphate MeSH
• DNA, Single-Stranded MeSH
• RAD51 protein, human MeSH Browser
• Rad51 Recombinase MeSH

Replication across damaged DNA templates is accompanied by transient formation of sister chromatid junctions (SCJs). Cells lacking Esc2, an adaptor protein containing no known enzymatic domains, are defective in the metabolism of these SCJs. However, how Esc2 is involved in the metabolism of SCJs remains elusive. Here we show interaction between Esc2 and a structure-specific endonuclease Mus81-Mms4 (the Mus81 complex), their involvement in the metabolism of SCJs, and the effects Esc2 has on the enzymatic activity of the Mus81 complex. We found that Esc2 specifically interacts with the Mus81 complex via its SUMO-like domains, stimulates enzymatic activity of the Mus81 complex in vitro, and is involved in the Mus81 complex-dependent resolution of SCJs in vivo Collectively, our data point to the possibility that the involvement of Esc2 in the metabolism of SCJs is, in part, via modulation of the activity of the Mus81 complex.

• MeSH
• Chromatids chemistry   metabolism   MeSH
• DNA, Fungal genetics   metabolism   MeSH
• DNA-Binding Proteins chemistry   genetics   metabolism   MeSH
• Endonucleases chemistry   genetics   metabolism   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Nuclear Proteins chemistry   genetics   metabolism   MeSH
• Cloning, Molecular MeSH
• DNA, Cruciform chemistry   metabolism   MeSH
• Small Ubiquitin-Related Modifier Proteins chemistry   genetics   metabolism   MeSH
• Genomic Instability MeSH
• DNA Damage MeSH
• Protein Domains MeSH
• Cell Cycle Proteins MeSH
• Gene Expression Regulation, Fungal * MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• DNA Replication MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Fungal MeSH
• DNA-Binding Proteins MeSH
• Endonucleases MeSH
• Esc2 protein, S cerevisiae MeSH Browser
• Nuclear Proteins MeSH
• DNA, Cruciform MeSH
• Small Ubiquitin-Related Modifier Proteins MeSH
• MUS81 protein, S cerevisiae MeSH Browser
• Cell Cycle Proteins MeSH
• Recombinant Proteins MeSH
• Saccharomyces cerevisiae Proteins MeSH

Central to homologous recombination in eukaryotes is the RAD51 recombinase, which forms helical nucleoprotein filaments on single-stranded DNA (ssDNA) and catalyzes strand invasion with homologous duplex DNA. Various regulatory proteins assist this reaction including the RAD51 paralogs. We recently discovered that a RAD51 paralog complex from C. elegans, RFS-1/RIP-1, functions predominantly downstream of filament assembly by binding and remodeling RAD-51-ssDNA filaments to a conformation more proficient for strand exchange. Here, we demonstrate that RFS-1/RIP-1 acts by shutting down RAD-51 dissociation from ssDNA. Using stopped-flow experiments, we show that RFS-1/RIP-1 confers this dramatic stabilization by capping the 5' end of RAD-51-ssDNA filaments. Filament end capping propagates a stabilizing effect with a 5'→3' polarity approximately 40 nucleotides along individual filaments. Finally, we discover that filament capping and stabilization are dependent on nucleotide binding, but not hydrolysis by RFS-1/RIP-1. These data define the mechanism of RAD51 filament remodeling by RAD51 paralogs.

• Keywords
• DNA repair, Rad51, Rad51 paralogs, filaments, genome stability, homologous recombination,
• MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• Intermediate Filaments genetics   metabolism   MeSH
• DNA, Single-Stranded genetics   MeSH
• Multiprotein Complexes metabolism   MeSH
• Caenorhabditis elegans Proteins genetics   metabolism   MeSH
• Recombinational DNA Repair MeSH
• Rad51 Recombinase genetics   metabolism   MeSH
• Carrier Proteins genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• DNA, Single-Stranded MeSH
• Multiprotein Complexes MeSH
• Caenorhabditis elegans Proteins MeSH
• rad-51 protein, C elegans MeSH Browser
• Rad51 Recombinase MeSH
• RFS-1 protein, C elegans MeSH Browser
• RIP-1 protein, C elegans MeSH Browser
• Carrier Proteins MeSH

Oncogene-evoked replication stress (RS) fuels genomic instability in diverse cancer types. Here we report that BRCA1, traditionally regarded a tumour suppressor, plays an unexpected tumour-promoting role in glioblastoma (GBM), safeguarding a protective response to supraphysiological RS levels. Higher BRCA1 positivity is associated with shorter survival of glioma patients and the abrogation of BRCA1 function in GBM enhances RS, DNA damage (DD) accumulation and impairs tumour growth. Mechanistically, we identify a novel role of BRCA1 as a transcriptional co-activator of RRM2 (catalytic subunit of ribonucleotide reductase), whereby BRCA1-mediated RRM2 expression protects GBM cells from endogenous RS, DD and apoptosis. Notably, we show that treatment with a RRM2 inhibitor triapine reproduces the BRCA1-depletion GBM-repressive phenotypes and sensitizes GBM cells to PARP inhibition. We propose that GBM cells are addicted to the RS-protective role of the BRCA1-RRM2 axis, targeting of which may represent a novel paradigm for therapeutic intervention in GBM.

• MeSH
• Survival Analysis MeSH
• Glioblastoma genetics   metabolism   pathology   MeSH
• Carcinogenesis genetics   MeSH
• Humans MeSH
• Mice, Inbred BALB C MeSH
• Mice, Nude MeSH
• Cell Line, Tumor MeSH
• Tumor Cells, Cultured MeSH
• Brain Neoplasms genetics   metabolism   pathology   MeSH
• BRCA1 Protein genetics   metabolism   MeSH
• Gene Expression Regulation, Neoplastic * MeSH
• DNA Replication genetics   MeSH
• Retrospective Studies MeSH
• Ribonucleoside Diphosphate Reductase genetics   metabolism   MeSH
• RNA Interference MeSH
• Transplantation, Heterologous MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BRCA1 protein, human MeSH Browser
• BRCA1 Protein MeSH
• ribonucleotide reductase M2 MeSH Browser
• Ribonucleoside Diphosphate Reductase MeSH