MRE11 nuclease is a central player in signaling and processing DNA damage, and in resolving stalled replication forks. Here, we describe the identification and characterization of new MRE11 inhibitors MU147 and MU1409. Both compounds inhibit MRE11 nuclease more specifically and effectively than the relatively weak state-of-the-art inhibitor mirin. They also abrogate double-strand break repair mechanisms that rely on MRE11 nuclease activity, without impairing ATM activation. Inhibition of MRE11 also impairs nascent strand degradation of stalled replication forks and selectively affects BRCA2-deficient cells. Herein, we illustrate that our newly discovered compounds MU147 and MU1409 can be used as chemical probes to further explore the biological role of MRE11 and support the potential clinical relevance of pharmacological inhibition of this nuclease.
- MeSH
- homologní protein MRE11 * metabolismus antagonisté a inhibitory MeSH
- inhibitory enzymů * farmakologie chemie chemická syntéza MeSH
- lidé MeSH
- molekulární struktura MeSH
- objevování léků MeSH
- oprava DNA účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation. In Rad54 a so-called extended region adjacent to motif III is involved in ATPase activity. Although the EcoR124I extended region bears sequence and structural similarities with Rad54, it does not influence ATPase or restriction activity as shown in this work, but mutagenesis of the conserved glycine residue of its motif III does alter ATPase and DNA cleavage activity. Through the lens of molecular dynamics, a full model of HsdR of EcoR124I based on available crystal structures allowed interpretation of functional effects of mutants in motif III and its extended region. The results indicate that the conserved glycine residue of motif III has a role in positioning the two helicase domains.
- MeSH
- adenosintrifosfát chemie MeSH
- aktivace enzymů MeSH
- analýza hlavních komponent MeSH
- DNA-helikasy chemie genetika metabolismus MeSH
- hydrolýza MeSH
- interakční proteinové domény a motivy * MeSH
- konformace proteinů MeSH
- multienzymové komplexy chemie MeSH
- mutace MeSH
- podjednotky proteinů chemie genetika metabolismus MeSH
- restrikční endonukleasy typu I chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Proper DNA replication is essential for faithful transmission of the genome. However, replication stress has serious impact on the integrity of the cell, leading to stalling or collapse of replication forks, and has been determined as a driving force of carcinogenesis. Mus81-Mms4 complex is a structure-specific endonuclease previously shown to be involved in processing of aberrant replication intermediates and promotes POLD3-dependent DNA synthesis via break-induced replication. However, how replication components might be involved in this process is not known. RESULTS: Herein, we show the interaction and robust stimulation of Mus81-Mms4 nuclease activity by heteropentameric replication factor C (RFC) complex, the processivity factor of replicative DNA polymerases that is responsible for loading of proliferating cell nuclear antigen (PCNA) during DNA replication and repair. This stimulation is enhanced by RFC-dependent ATP hydrolysis and by PCNA loading on the DNA. Moreover, this stimulation is not specific to Rfc1, the largest of subunit of this complex, thus indicating that alternative clamp loaders may also play a role in the stimulation. We also observed a targeting of Mus81 by RFC to the nick-containing DNA substrate and we provide further evidence that indicates cooperation between Mus81 and the RFC complex in the repair of DNA lesions generated by various DNA-damaging agents. CONCLUSIONS: Identification of new interacting partners and modulators of Mus81-Mms4 nuclease, RFC, and PCNA imply the cooperation of these factors in resolution of stalled replication forks and branched DNA structures emanating from the restarted replication forks under conditions of replication stress.
- MeSH
- "flap" endonukleasy genetika metabolismus MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- endonukleasy genetika metabolismus MeSH
- proliferační antigen buněčného jádra genetika metabolismus MeSH
- rekombinace genetická MeSH
- replikace DNA MeSH
- replikační protein C genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Accurate completion of replication relies on the ability of cells to activate error-free recombination-mediated DNA damage bypass at sites of perturbed replication. However, as anti-recombinase activities are also recruited to replication forks, how recombination-mediated damage bypass is enabled at replication stress sites remained puzzling. Here we uncovered that the conserved SUMO-like domain-containing Saccharomyces cerevisiae protein Esc2 facilitates recombination-mediated DNA damage tolerance by allowing optimal recruitment of the Rad51 recombinase specifically at sites of perturbed replication. Mechanistically, Esc2 binds stalled replication forks and counteracts the anti-recombinase Srs2 helicase via a two-faceted mechanism involving chromatin recruitment and turnover of Srs2. Importantly, point mutations in the SUMO-like domains of Esc2 that reduce its interaction with Srs2 cause suboptimal levels of Rad51 recruitment at damaged replication forks. In conclusion, our results reveal how recombination-mediated DNA damage tolerance is locally enabled at sites of replication stress and globally prevented at undamaged replicating chromosomes.
- MeSH
- bodová mutace MeSH
- chromatin metabolismus MeSH
- DNA-helikasy genetika metabolismus MeSH
- jaderné proteiny genetika metabolismus MeSH
- poškození DNA genetika MeSH
- rekombinace genetická genetika MeSH
- rekombinasa Rad51 metabolismus MeSH
- replikace DNA genetika MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae enzymologie genetika MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability.
- MeSH
- DNA-helikasy genetika metabolismus MeSH
- DNA-polymerasa II genetika metabolismus MeSH
- DNA-polymerasa III genetika metabolismus MeSH
- homologní rekombinace * MeSH
- mutace genetika MeSH
- nestabilita genomu MeSH
- oprava DNA genetika účinky záření MeSH
- poškození DNA genetika účinky záření MeSH
- proliferační antigen buněčného jádra genetika metabolismus MeSH
- protein SUMO-1 genetika metabolismus MeSH
- rekombinasa Rad51 genetika metabolismus MeSH
- replikace DNA genetika účinky záření MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sumoylace MeSH
- ultrafialové záření škodlivé účinky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
