Homologous recombination (HR) factors are crucial for DSB repair and processing stalled replication forks. RAD51 paralogs, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, have emerged as essential tumour suppressors, forming two subcomplexes, BCDX2 and CX3. Mutations in these genes are associated with cancer susceptibility and Fanconi anaemia, yet their biochemical activities remain unclear. This study reveals a linear arrangement of BCDX2 subunits compared to the RAD51 ring. BCDX2 shows a strong affinity towards single-stranded DNA (ssDNA) via unique binding mechanism compared to RAD51, and a contribution of DX2 subunits in binding branched DNA substrates. We demonstrate that BCDX2 facilitates RAD51 loading on ssDNA by suppressing the cooperative requirement of RAD51 binding to DNA and stabilizing the filament. Notably, BCDX2 also promotes RAD51 loading on short ssDNA and reversed replication fork substrates. Moreover, while mutants defective in ssDNA binding retain the ability to bind branched DNA substrates, they still facilitate RAD51 loading onto reversed replication forks. Our study provides mechanistic insights into how the BCDX2 complex stimulates the formation of BRCA2-independent RAD51 filaments on short stretches of ssDNA present at ssDNA gaps or stalled replication forks, highlighting its role in genome maintenance and DNA repair.

• MeSH
• DNA vazebné proteiny * metabolismus   genetika   MeSH
• jednovláknová DNA * metabolismus   genetika   MeSH
• lidé MeSH
• multiproteinové komplexy MeSH
• mutace MeSH
• rekombinasa Rad51 * metabolismus   genetika   MeSH
• replikace DNA * genetika   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny * MeSH
• jednovláknová DNA * MeSH
• multiproteinové komplexy MeSH
• RAD51 protein, human MeSH Prohlížeč
• rekombinasa Rad51 * MeSH

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
• adenosindifosfát farmakologie   MeSH
• adenosintrifosfát farmakologie   MeSH
• Caenorhabditis elegans metabolismus   MeSH
• druhová specificita MeSH
• fluorescence MeSH
• interferometrie MeSH
• jednovláknová DNA metabolismus   MeSH
• nukleotidy metabolismus   MeSH
• proteiny Caenorhabditis elegans metabolismus   MeSH
• rekombinasa Rad51 chemie   metabolismus   MeSH
• sekvenční homologie aminokyselin * MeSH
• stabilita proteinů účinky léků   MeSH
• vazba proteinů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosindifosfát MeSH
• adenosintrifosfát MeSH
• jednovláknová DNA MeSH
• nukleotidy MeSH
• proteiny Caenorhabditis elegans MeSH
• rekombinasa Rad51 MeSH

Extracellular pH has been assumed to play little if any role in how bacteria respond to antibiotics and antibiotic resistance development. Here, we show that the intracellular pH of Escherichia coli equilibrates to the environmental pH following treatment with the DNA damaging antibiotic nalidixic acid. We demonstrate that this allows the environmental pH to influence the transcription of various DNA damage response genes and physiological processes such as filamentation. Using purified RecA and a known pH-sensitive mutant variant RecA K250R we show how pH can affect the biochemical activity of a protein central to control of the bacterial DNA damage response system. Finally, two different mutagenesis assays indicate that environmental pH affects antibiotic resistance development. Specifically, at environmental pH's greater than six we find that mutagenesis plays a significant role in producing antibiotic resistant mutants. At pH's less than or equal to 6 the genome appears more stable but extensive filamentation is observed, a phenomenon that has previously been linked to increased survival in the presence of macrophages.

• MeSH
• antibakteriální látky farmakologie   MeSH
• Escherichia coli účinky léků   genetika   účinky záření   MeSH
• koncentrace vodíkových iontů MeSH
• kyselina nalidixová farmakologie   MeSH
• mikrobiální viabilita účinky léků   účinky záření   MeSH
• nestabilita genomu účinky léků   genetika   účinky záření   MeSH
• poškození DNA účinky léků   genetika   účinky záření   MeSH
• propidium farmakologie   MeSH
• průtoková cytometrie MeSH
• retardační test MeSH
• rifampin farmakologie   MeSH
• ultrafialové záření MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• kyselina nalidixová MeSH
• propidium MeSH
• rifampin MeSH

The proper repair of deleterious DNA lesions such as double strand breaks prevents genomic instability and carcinogenesis. In yeast, the Rad52 protein mediates DSB repair via homologous recombination. In mammalian cells, despite the presence of the RAD52 protein, the tumour suppressor protein BRCA2 acts as the predominant mediator during homologous recombination. For decades, it has been believed that the RAD52 protein played only a back-up role in the repair of DSBs performing an error-prone single strand annealing (SSA). Recent studies have identified several new functions of the RAD52 protein and have drawn attention to its important role in genome maintenance. Here, we show that RAD52 activities are enhanced by interacting with a small and highly acidic protein called DSS1. Binding of DSS1 to RAD52 changes the RAD52 oligomeric conformation, modulates its DNA binding properties, stimulates SSA activity and promotes strand invasion. Our work introduces for the first time RAD52 as another interacting partner of DSS1 and shows that both proteins are important players in the SSA and BIR pathways of DSB repair.

• MeSH
• DNA opravný a rekombinační protein Rad52 genetika   MeSH
• DNA vazebné proteiny genetika   MeSH
• dvouřetězcové zlomy DNA MeSH
• genom lidský genetika   MeSH
• homologní rekombinace genetika   MeSH
• karcinogeneze genetika   MeSH
• lidé MeSH
• nestabilita genomu genetika   MeSH
• oprava DNA genetika   MeSH
• osteosarkom genetika   patologie   MeSH
• proteasomový endopeptidasový komplex genetika   MeSH
• protein BRCA2 genetika   MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• BRCA2 protein, human MeSH Prohlížeč
• DNA opravný a rekombinační protein Rad52 MeSH
• DNA vazebné proteiny MeSH
• proteasomový endopeptidasový komplex MeSH
• protein BRCA2 MeSH
• RAD52 protein, human MeSH Prohlížeč
• RAD52 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• SEM1 protein, human MeSH Prohlížeč

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
• adeninnukleotidy metabolismus   MeSH
• adenosintrifosfát metabolismus   MeSH
• biologická evoluce MeSH
• elektronová kryomikroskopie MeSH
• jednovláknová DNA metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• molekulární modely MeSH
• mutace MeSH
• rekombinasa Rad51 genetika   metabolismus   ultrastruktura   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adeninnukleotidy MeSH
• adenosintrifosfát MeSH
• jednovláknová DNA MeSH
• RAD51 protein, human MeSH Prohlížeč
• rekombinasa Rad51 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.

• Klíčová slova
• DNA repair, Rad51, Rad51 paralogs, filaments, genome stability, homologous recombination,
• MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• intermediární filamenta genetika   metabolismus   MeSH
• jednovláknová DNA genetika   MeSH
• multiproteinové komplexy metabolismus   MeSH
• proteiny Caenorhabditis elegans genetika   metabolismus   MeSH
• rekombinační oprava DNA MeSH
• rekombinasa Rad51 genetika   metabolismus   MeSH
• transportní proteiny genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• jednovláknová DNA MeSH
• multiproteinové komplexy MeSH
• proteiny Caenorhabditis elegans MeSH
• rad-51 protein, C elegans MeSH Prohlížeč
• rekombinasa Rad51 MeSH
• RFS-1 protein, C elegans MeSH Prohlížeč
• RIP-1 protein, C elegans MeSH Prohlížeč
• transportní proteiny MeSH