Homologous recombination (HR) protects replication forks (RFs) and repairs DNA double-strand breaks (DSBs). Within HR, BRCA2 regulates RAD51 via two interaction regions: the BRC repeats to form filaments on single-stranded DNA and exon 27 (Ex27) to stabilize the filament. Here, we identified a RAD51 S181P mutant that selectively disrupted the RAD51-Ex27 association while maintaining interaction with BRC repeat and proficiently forming filaments capable of DNA binding and strand invasion. Interestingly, RAD51 S181P was defective for RF protection/restart but proficient for DSB repair. Our data suggest that Ex27-mediated stabilization of RAD51 filaments is required for the protection of RFs, while it seems dispensable for the repair of DSBs.

• Klíčová slova
• Genetics, Molecular biology, Molecular interaction, Properties of biomolecules,
• Publikační typ
• časopisecké články MeSH

DNA damage repair (DDR) is a safeguard for genome integrity maintenance. Increasing DDR efficiency could increase the yield of induced pluripotent stem cells (iPSC) upon reprogramming from somatic cells. The epigenetic mechanisms governing DDR during iPSC reprogramming are not completely understood. Our goal was to evaluate the splicing isoforms of histone variant macroH2A1, macroH2A1.1, and macroH2A1.2, as potential regulators of DDR during iPSC reprogramming. GFP-Trap one-step isolation of mtagGFP-macroH2A1.1 or mtagGFP-macroH2A1.2 fusion proteins from overexpressing human cell lines, followed by liquid chromatography-tandem mass spectrometry analysis, uncovered macroH2A1.1 exclusive interaction with Poly-ADP Ribose Polymerase 1 (PARP1) and X-ray cross-complementing protein 1 (XRCC1). MacroH2A1.1 overexpression in U2OS-GFP reporter cells enhanced specifically nonhomologous end joining (NHEJ) repair pathway, while macroH2A1.1 knock-out (KO) mice showed an impaired DDR capacity. The exclusive interaction of macroH2A1.1, but not macroH2A1.2, with PARP1/XRCC1, was confirmed in human umbilical vein endothelial cells (HUVEC) undergoing reprogramming into iPSC through episomal vectors. In HUVEC, macroH2A1.1 overexpression activated transcriptional programs that enhanced DDR and reprogramming. Consistently, macroH2A1.1 but not macroH2A1.2 overexpression improved iPSC reprogramming. We propose the macroH2A1 splicing isoform macroH2A1.1 as a promising epigenetic target to improve iPSC genome stability and therapeutic potential.

• Klíčová slova
• DNA damage, cell reprogramming, induced pluripotent stem cells, macroH2A1.1,
• MeSH
• DNA MeSH
• endoteliální buňky metabolismus   MeSH
• histony * metabolismus   MeSH
• indukované pluripotentní kmenové buňky * metabolismus   MeSH
• lidé MeSH
• myši MeSH
• oprava DNA MeSH
• protein XRCC1 genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH
• histony * MeSH
• MACROH2A1 protein, human MeSH Prohlížeč
• protein XRCC1 MeSH
• XRCC1 protein, human MeSH Prohlížeč

Genotoxic stress triggers a combined action of DNA repair and cell cycle checkpoint pathways. Protein phosphatase 2C delta (referred to as WIP1) is involved in timely inactivation of DNA damage response by suppressing function of p53 and other targets at chromatin. Here we show that WIP1 promotes DNA repair through homologous recombination. Loss or inhibition of WIP1 delayed disappearance of the ionizing radiation-induced 53BP1 foci in S/G2 cells and promoted cell death. We identify breast cancer associated protein 1 (BRCA1) as interactor and substrate of WIP1 and demonstrate that WIP1 activity is needed for correct dynamics of BRCA1 recruitment to chromatin flanking the DNA lesion. In addition, WIP1 dephosphorylates 53BP1 at Threonine 543 that was previously implicated in mediating interaction with RIF1. Finally, we report that inhibition of WIP1 allowed accumulation of DNA damage in S/G2 cells and increased sensitivity of cancer cells to a poly-(ADP-ribose) polymerase inhibitor olaparib. We propose that inhibition of WIP1 may increase sensitivity of BRCA1-proficient cancer cells to olaparib.

• Klíčová slova
• DNA repair, PARP inhibitor, chemotherapy, genotoxic stress, olaparib, phosphatase,
• MeSH
• 53BP1 metabolismus   MeSH
• antitumorózní látky farmakologie   MeSH
• apoptóza účinky léků   MeSH
• chemorezistence účinky léků   MeSH
• chromatin metabolismus   MeSH
• ftalaziny farmakologie   MeSH
• HEK293 buňky MeSH
• homologní rekombinace genetika   MeSH
• kontrolní body fáze G2 buněčného cyklu MeSH
• kontrolní body fáze S buněčného cyklu MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prsu metabolismus   MeSH
• oprava DNA genetika   fyziologie   MeSH
• PARP inhibitory farmakologie   MeSH
• piperaziny farmakologie   MeSH
• poškození DNA genetika   fyziologie   MeSH
• proliferace buněk účinky léků   MeSH
• protein BRCA1 metabolismus   MeSH
• proteinfosfatasa 2C antagonisté a inhibitory   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 53BP1 MeSH
• antitumorózní látky MeSH
• BRCA1 protein, human MeSH Prohlížeč
• chromatin MeSH
• ftalaziny MeSH
• olaparib MeSH Prohlížeč
• PARP inhibitory MeSH
• piperaziny MeSH
• PPM1D protein, human MeSH Prohlížeč
• protein BRCA1 MeSH
• proteinfosfatasa 2C MeSH
• TP53BP1 protein, human MeSH Prohlížeč

The MRN (MRE11-RAD50-NBS1) complex is essential for repair of DNA double-strand breaks and stalled replication forks. Mutations of the MRN complex subunit MRE11 cause the hereditary cancer-susceptibility disease ataxia-telangiectasia-like disorder (ATLD). Here we show that MRE11 directly interacts with PIH1D1, a subunit of heat-shock protein 90 cochaperone R2TP complex, which is required for the assembly of large protein complexes, such as RNA polymerase II, small nucleolar ribonucleoproteins and mammalian target of rapamycin complex 1. The MRE11-PIH1D1 interaction is dependent on casein kinase 2 (CK2) phosphorylation of two acidic sequences within the MRE11 C terminus containing serines 558/561 and 688/689. Conversely, the PIH1D1 phospho-binding domain PIH-N is required for association with MRE11 phosphorylated by CK2. Consistent with these findings, depletion of PIH1D1 resulted in MRE11 destabilization and affected DNA-damage repair processes dependent on MRE11. Additionally, mutations of serines 688/689, which abolish PIH1D1 binding, also resulted in decreased MRE11 stability. As depletion of R2TP frequently leads to instability of its substrates and as truncation mutation of MRE11 lacking serines 688/689 leads to decreased levels of the MRN complex both in ATLD patients and an ATLD mouse model, our results suggest that the MRN complex is a novel R2TP complex substrate and that their interaction is regulated by CK2 phosphorylation.

• MeSH
• ATM protein metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• enzymy opravy DNA metabolismus   MeSH
• fosforylace fyziologie   MeSH
• jaderné proteiny metabolismus   MeSH
• kaseinkinasa II metabolismus   MeSH
• lidé MeSH
• mutace fyziologie   MeSH
• myši MeSH
• oprava DNA fyziologie   MeSH
• poškození DNA fyziologie   MeSH
• proteiny regulující apoptózu metabolismus   MeSH
• proteiny tepelného šoku metabolismus   MeSH
• ribonukleoproteiny malé jadérkové metabolismus   MeSH
• RNA-polymerasa II metabolismus   MeSH
• serin metabolismus   MeSH
• teleangiektatická ataxie metabolismus   MeSH
• TOR serin-threoninkinasy metabolismus   MeSH
• vazba proteinů fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ATM protein MeSH
• DNA vazebné proteiny MeSH
• enzymy opravy DNA MeSH
• jaderné proteiny MeSH
• kaseinkinasa II MeSH
• proteiny regulující apoptózu MeSH
• proteiny tepelného šoku MeSH
• ribonukleoproteiny malé jadérkové MeSH
• RNA-polymerasa II MeSH
• serin MeSH
• TOR serin-threoninkinasy MeSH

The 5'-3' resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5'-3' DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIIIα-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells.

• Klíčová slova
• DNA Damage, DNA Helicase, DNA Recombination, DNA Repair, Genomic Instability, RecQ,
• MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• DNA-helikasy genetika   metabolismus   MeSH
• DNA genetika   metabolismus   MeSH
• dvouřetězcové zlomy DNA * MeSH
• enzymy opravy DNA genetika   metabolismus   MeSH
• exodeoxyribonukleasy genetika   metabolismus   MeSH
• genetická epistáze fyziologie   MeSH
• HEK293 buňky MeSH
• helikasy RecQ genetika   metabolismus   MeSH
• helikáza Wernerova syndromu MeSH
• homologní protein MRE11 MeSH
• hydrolasy působící na anhydridy kyselin MeSH
• lidé MeSH
• multienzymové komplexy genetika   metabolismus   MeSH
• ubikvitin aktivující enzymy genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Bloom syndrome protein MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• DNA-helikasy MeSH
• DNA MeSH
• DNA2 protein, human MeSH Prohlížeč
• enzymy opravy DNA MeSH
• exodeoxyribonukleasy MeSH
• helikasy RecQ MeSH
• helikáza Wernerova syndromu MeSH
• homologní protein MRE11 MeSH
• hydrolasy působící na anhydridy kyselin MeSH
• MRE11 protein, human MeSH Prohlížeč
• multienzymové komplexy MeSH
• RAD50 protein, human MeSH Prohlížeč
• UBA2 protein, human MeSH Prohlížeč
• ubikvitin aktivující enzymy MeSH
• WRN protein, human MeSH Prohlížeč

Most mitotic homologous recombination (HR) events proceed via a synthesis-dependent strand annealing mechanism to avoid crossing over, which may give rise to chromosomal rearrangements and loss of heterozygosity. The molecular mechanisms controlling HR sub-pathway choice are poorly understood. Here, we show that human RECQ5, a DNA helicase that can disrupt RAD51 nucleoprotein filaments, promotes formation of non-crossover products during DNA double-strand break-induced HR and counteracts the inhibitory effect of RAD51 on RAD52-mediated DNA annealing in vitro and in vivo. Moreover, we demonstrate that RECQ5 deficiency is associated with an increased occupancy of RAD51 at a double-strand break site, and it also causes an elevation of sister chromatid exchanges on inactivation of the Holliday junction dissolution pathway or on induction of a high load of DNA damage in the cell. Collectively, our findings suggest that RECQ5 acts during the post-synaptic phase of synthesis-dependent strand annealing to prevent formation of aberrant RAD51 filaments on the extended invading strand, thus limiting its channeling into potentially hazardous crossover pathway of HR.

• MeSH
• buněčné linie MeSH
• DNA opravný a rekombinační protein Rad52 metabolismus   MeSH
• DNA metabolismus   MeSH
• dvouřetězcové zlomy DNA * MeSH
• helikasy RecQ metabolismus   MeSH
• jednovláknová DNA metabolismus   MeSH
• lidé MeSH
• rekombinační oprava DNA * MeSH
• rekombinasa Rad51 metabolismus   MeSH
• výměna sesterských chromatid MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA opravný a rekombinační protein Rad52 MeSH
• DNA MeSH
• helikasy RecQ MeSH
• jednovláknová DNA MeSH
• RECQL5 protein, human MeSH Prohlížeč
• rekombinasa Rad51 MeSH