During homologous recombination, Dbl2 protein is required for localisation of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments. RNA-seq analysis of dbl2Δ transcriptome showed that the dbl2 deletion results in upregulation of more than 500 loci in Schizosaccharomyces pombe. Compared with the loci with no change in expression, the misregulated loci in dbl2Δ are closer to long terminal and long tandem repeats. Furthermore, the misregulated loci overlap with antisense transcripts, retrotransposons, meiotic genes and genes located in subtelomeric regions. A comparison of the expression profiles revealed that Dbl2 represses the same type of genes as the HIRA histone chaperone complex. Although dbl2 deletion does not alleviate centromeric or telomeric silencing, it suppresses the silencing defect at the outer centromere caused by deletion of hip1 and slm9 genes encoding subunits of the HIRA complex. Moreover, our analyses revealed that cells lacking dbl2 show a slight increase of nucleosomes at transcription start sites and increased levels of methylated histone H3 (H3K9me2) at centromeres, subtelomeres, rDNA regions and long terminal repeats. Finally, we show that other proteins involved in homologous recombination, such as Fbh1, Rad51, Mus81 and Rad54, participate in the same gene repression pathway.

• MeSH
• centromera MeSH
• histonový kód MeSH
• homologní rekombinace * MeSH
• nukleozomy metabolismus   MeSH
• proteiny buněčného cyklu antagonisté a inhibitory   metabolismus   MeSH
• regulace genové exprese u hub * MeSH
• represorové proteiny fyziologie   MeSH
• Schizosaccharomyces pombe - proteiny antagonisté a inhibitory   metabolismus   fyziologie   MeSH
• Schizosaccharomyces genetika   MeSH
• transkripční faktory antagonisté a inhibitory   metabolismus   MeSH
• umlčování genů * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Dbl2 protein, S pombe MeSH Prohlížeč
• hip1 protein, S pombe MeSH Prohlížeč
• nukleozomy MeSH
• proteiny buněčného cyklu MeSH
• represorové proteiny MeSH
• Schizosaccharomyces pombe - proteiny MeSH
• Slm9 protein, S pombe MeSH Prohlížeč
• transkripční faktory MeSH

Nuclear architecture plays a significant role in DNA repair mechanisms. It is evident that proteins involved in DNA repair are compartmentalized in not only spontaneously occurring DNA lesions or ionizing radiation-induced foci (IRIF), but a specific clustering of these proteins can also be observed within the whole cell nucleus. For example, 53BP1-positive and BRCA1-positive DNA repair foci decorate chromocenters and can appear close to nuclear speckles. Both 53BP1 and BRCA1 are well-described factors that play an essential role in double-strand break (DSB) repair. These proteins are members of two protein complexes: 53BP1-RIF1-PTIP and BRCA1-CtIP, which make a "decision" determining whether canonical nonhomologous end joining (NHEJ) or homology-directed repair (HDR) is activated. It is generally accepted that 53BP1 mediates the NHEJ mechanism, while HDR is activated via a BRCA1-dependent signaling pathway. Interestingly, the 53BP1 protein appears relatively quickly at DSB sites, while BRCA1 is functional at later stages of DNA repair, as soon as the Mre11-Rad50-Nbs1 complex is recruited to the DNA lesions. A function of the 53BP1 protein is also linked to a specific histone signature, including phosphorylation of histone H2AX (γH2AX) or methylation of histone H4 at the lysine 20 position (H4K20me); therefore, we also discuss an epigenetic landscape of 53BP1-positive DNA lesions.

• Klíčová slova
• 53BP1, BRCA1, DNA damage, epigenetics, histone modifications,
• MeSH
• 53BP1 genetika   metabolismus   MeSH
• buněčné jádro genetika   metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• oprava DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• 53BP1 MeSH

A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81-Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ssDNA gaps together with Mus81-Mms4. In this study, we show that the Srs2 and Mus81-Mms4 proteins physically interact in vitro and in vivo and we map the interaction domains within the Srs2 and Mus81 proteins. Further, we show that Srs2 plays a dual role in the stimulation of the Mus81-Mms4 nuclease activity on a variety of DNA substrates. First, Srs2 directly stimulates Mus81-Mms4 nuclease activity independent of its helicase activity. Second, Srs2 removes Rad51 from DNA to allow access of Mus81-Mms4 to cleave DNA. Concomitantly, Mus81-Mms4 inhibits the helicase activity of Srs2. Taken together, our data point to a coordinated role of Mus81-Mms4 and Srs2 in processing of recombination as well as replication intermediates.

• MeSH
• "flap" endonukleasy fyziologie   MeSH
• DNA primery MeSH
• DNA vazebné proteiny fyziologie   MeSH
• DNA-helikasy fyziologie   MeSH
• endonukleasy fyziologie   MeSH
• fluorescenční mikroskopie MeSH
• polymerázová řetězová reakce MeSH
• rekombinace genetická * MeSH
• Saccharomyces cerevisiae - proteiny fyziologie   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sekvence nukleotidů MeSH
• techniky dvojhybridového systému MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• "flap" endonukleasy MeSH
• DNA primery MeSH
• DNA vazebné proteiny MeSH
• DNA-helikasy MeSH
• endonukleasy MeSH
• MMS4 protein, S cerevisiae MeSH Prohlížeč
• MUS81 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• SRS2 protein, S cerevisiae MeSH Prohlížeč

Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology-dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock-out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B-dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double-stranded breaks (measured as γ-H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S-phase, and is ATM-independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non-transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double-stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single-stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability.

• Klíčová slova
• 45S rDNA, Arabidopsis thaliana, DNA repair, FAS1, FAS2, RAD51B, chromatin assembly factor 1, genome instability,
• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• faktor 1 pro uspořádání chromatinu genetika   metabolismus   MeSH
• nestabilita genomu genetika   fyziologie   MeSH
• oprava DNA genetika   fyziologie   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• ribozomální DNA genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• faktor 1 pro uspořádání chromatinu MeSH
• FAS protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• RAD51B protein, Arabidopsis MeSH Prohlížeč
• ribozomální DNA MeSH

DNA repair scaffolds mediate specific DNA and protein interactions in order to assist repair enzymes in recognizing and removing damaged sequences. Many scaffold proteins are dedicated to repairing a particular type of lesion. Here, we show that the budding yeast Saw1 scaffold is more versatile. It helps cells cope with base lesions and protein-DNA adducts through its known function of recruiting the Rad1-Rad10 nuclease to DNA. In addition, it promotes UV survival via a mechanism mediated by its sumoylation. Saw1 sumoylation favors its interaction with another nuclease Slx1-Slx4, and this SUMO-mediated role is genetically separable from two main UV lesion repair processes. These effects of Saw1 and its sumoylation suggest that Saw1 is a multifunctional scaffold that can facilitate diverse types of DNA repair through its modification and nuclease interactions.

• MeSH
• analýza přežití MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• endonukleasy genetika   metabolismus   MeSH
• oprava DNA * MeSH
• poškození DNA * MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   genetika   metabolismus   MeSH
• sumoylace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• endonukleasy MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• Saw1 protein, S cerevisiae MeSH Prohlížeč

The Saccharomyces cerevisiae Rad1-Rad10 complex is a conserved, structure-specific endonuclease important for repairing multiple types of DNA lesions. Upon recruitment to lesion sites, Rad1-Rad10 removes damaged sequences, enabling subsequent gap filling and ligation. Acting at mid-steps of repair, the association and dissociation of Rad1-Rad10 with DNA can influence repair efficiency. We show that genotoxin-enhanced Rad1 sumoylation occurs after the nuclease is recruited to lesion sites. A single lysine outside Rad1's nuclease and Rad10-binding domains is sumoylated in vivo and in vitro. Mutation of this site to arginine abolishes Rad1 sumoylation and impairs Rad1-mediated repair at high doses of DNA damage, but sustains the repair of a single double-stranded break. The timing of Rad1 sumoylation and the phenotype bias toward high lesion loads point to a post-incision role for sumoylation, possibly affecting Rad1 dissociation from DNA. Indeed, biochemical examination shows that sumoylation of Rad1 decreases the complex's affinity for DNA without affecting other protein properties. These findings suggest a model whereby sumoylation of Rad1 promotes its disengagement from DNA after nuclease cleavage, allowing it to efficiently attend to large numbers of DNA lesions.

• MeSH
• DNA metabolismus   MeSH
• endonukleasy chemie   genetika   metabolismus   MeSH
• enzymy opravy DNA chemie   genetika   metabolismus   MeSH
• intracelulární signální peptidy a proteiny fyziologie   MeSH
• lysin metabolismus   MeSH
• mutace MeSH
• oprava DNA * MeSH
• poškození DNA MeSH
• protein-serin-threoninkinasy fyziologie   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   fyziologie   MeSH
• sumoylace * MeSH
• ubikvitinligasy fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA MeSH
• endonukleasy MeSH
• enzymy opravy DNA MeSH
• intracelulární signální peptidy a proteiny MeSH
• lysin MeSH
• MEC1 protein, S cerevisiae MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH
• RAD1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• Siz1 protein, S cerevisiae MeSH Prohlížeč
• Siz2 protein, S cerevisiae MeSH Prohlížeč
• ubikvitinligasy MeSH

Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.

• MeSH
• adenosintrifosfatasy chemie   MeSH
• aminokyselinové motivy MeSH
• DNA primery metabolismus   MeSH
• DNA-helikasy chemie   metabolismus   MeSH
• DNA metabolismus   MeSH
• enzymy opravy DNA chemie   metabolismus   MeSH
• konzervovaná sekvence MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• mutace genetika   MeSH
• mutační analýza DNA MeSH
• mutantní proteiny metabolismus   MeSH
• nestabilita genomu MeSH
• oprava DNA * MeSH
• párování chromozomů MeSH
• poškození DNA MeSH
• proliferační antigen buněčného jádra metabolismus   MeSH
• rekombinace genetická * MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• DNA primery MeSH
• DNA-helikasy MeSH
• DNA MeSH
• enzymy opravy DNA MeSH
• mutantní proteiny MeSH
• proliferační antigen buněčného jádra MeSH
• RAD54 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH

The budding yeast Srs2 protein possesses 3' to 5' DNA helicase activity and channels untimely recombination to post-replication repair by removing Rad51 from ssDNA. However, it also promotes recombination via a synthesis-dependent strand-annealing pathway (SDSA). Furthermore, at the replication fork, Srs2 is required for fork progression and prevents the instability of trinucleotide repeats. To better understand the multiple roles of the Srs2 helicase during these processes, we analysed the ability of Srs2 to bind and unwind various DNA substrates that mimic structures present during DNA replication and recombination. While leading or lagging strands were efficiently unwound, the presence of ssDNA binding protein RPA presented an obstacle for Srs2 translocation. We also tested the preferred directionality of unwinding of various substrates and studied the effect of Rad51 and Mre11 proteins on Srs2 helicase activity. These biochemical results help us understand the possible role of Srs2 in the processing of stalled or blocked replication forks as a part of post-replication repair as well as homologous recombination (HR).

• MeSH
• delece genu MeSH
• DNA-helikasy genetika   metabolismus   MeSH
• endodeoxyribonukleasy metabolismus   MeSH
• exodeoxyribonukleasy metabolismus   MeSH
• homologní rekombinace * MeSH
• jednovláknová DNA chemie   metabolismus   MeSH
• křížová struktura DNA chemie   metabolismus   MeSH
• rekombinasa Rad51 metabolismus   MeSH
• replikace DNA * MeSH
• replikační protein A metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-helikasy MeSH
• endodeoxyribonukleasy MeSH
• exodeoxyribonukleasy MeSH
• jednovláknová DNA MeSH
• křížová struktura DNA MeSH
• MRE11 protein, S cerevisiae MeSH Prohlížeč
• RAD51 protein, S cerevisiae MeSH Prohlížeč
• rekombinasa Rad51 MeSH
• replikační protein A MeSH
• RFA1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• SRS2 protein, S cerevisiae MeSH Prohlížeč

DNA double-strand breaks (DSBs) comprise one of the most toxic DNA lesions, as the failure to repair a single DSB has detrimental consequences on the cell. Homologous recombination (HR) constitutes an error-free repair pathway for the repair of DSBs. On the other hand, when uncontrolled, HR can lead to genome rearrangements and needs to be tightly regulated. In recent years, several proteins involved in different steps of HR have been shown to undergo modification by small ubiquitin-like modifier (SUMO) peptide and it has been suggested that deficient sumoylation impairs the progression of HR. This review addresses specific effects of sumoylation on the properties of various HR proteins and describes its importance for the homeostasis of DNA repetitive sequences. The article further illustrates the role of sumoylation in meiotic recombination and the interplay between SUMO and other post-translational modifications.

• Publikační typ
• časopisecké články MeSH

Homologous recombination (HR) is critical both for repairing DNA lesions in mitosis and for chromosomal pairing and exchange during meiosis. However, some forms of HR can also lead to undesirable DNA rearrangements. Multiple regulatory mechanisms have evolved to ensure that HR takes place at the right time, place and manner. Several of these impinge on the control of Rad51 nucleofilaments that play a central role in HR. Some factors promote the formation of these structures while others lead to their disassembly or the use of alternative repair pathways. In this article, we review these mechanisms in both mitotic and meiotic environments and in different eukaryotic taxa, with an emphasis on yeast and mammal systems. Since mutations in several proteins that regulate Rad51 nucleofilaments are associated with cancer and cancer-prone syndromes, we discuss how understanding their functions can lead to the development of better tools for cancer diagnosis and therapy.

• MeSH
• homologní rekombinace * MeSH
• lidé MeSH
• meióza MeSH
• nádory diagnóza   terapie   MeSH
• nemoc genetika   MeSH
• posttranslační úpravy proteinů MeSH
• rekombinasa Rad51 metabolismus   MeSH
• replikační protein A metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• rekombinasa Rad51 MeSH
• replikační protein A MeSH