DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-β-lactamase fold 5'-3' exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.
- MeSH
- DNA metabolismus genetika MeSH
- dvouřetězcové zlomy DNA * účinky záření MeSH
- enzymy opravy DNA * metabolismus genetika MeSH
- exodeoxyribonukleasy * metabolismus genetika MeSH
- lidé MeSH
- oprava DNA * MeSH
- proliferační antigen buněčného jádra metabolismus genetika MeSH
- proteiny buněčného cyklu MeSH
- ubikvitinace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Meiotic recombination is of central importance for the proper segregation of homologous chromosomes, but also for creating genetic diversity. It is initiated by the formation of double-strand breaks (DSBs) in DNA catalysed by evolutionarily conserved Spo11, together with additional protein partners. Difficulties in purifying the Spo11 protein have limited the characterization of its biochemical properties and of its interactions with other DSB proteins. In this study, we have purified fragments of Spo11 and show for the first time that Spo11 can physically interact with Mre11 and modulates its DNA binding, bridging, and nuclease activities. The interaction of Mre11 with Spo11 requires its far C-terminal region, which is in line with the severe meiotic phenotypes of various mre11 mutations located at the C-terminus. Moreover, calibrated ChIP for Mre11 shows that Spo11 promotes Mre11 recruitment to chromatin, independent of DSB formation. A mutant deficient in Spo11 interaction severely reduces the association of Mre11 with meiotic chromatin. Consistent with the reduction of Mre11 foci in this mutant, it strongly impedes DSB formation, leading to spore death. Our data provide evidence that physical interaction between Spo11 and Mre11, together with end-bridging, promote normal recruitment of Mre11 to hotspots and DSB formation.
- MeSH
- chromatin * metabolismus MeSH
- DNA vazebné proteiny metabolismus genetika MeSH
- dvouřetězcové zlomy DNA * MeSH
- endodeoxyribonukleasy * metabolismus genetika MeSH
- exodeoxyribonukleasy metabolismus genetika MeSH
- meióza * genetika MeSH
- mutace MeSH
- Saccharomyces cerevisiae - proteiny * metabolismus genetika MeSH
- Saccharomyces cerevisiae cytologie genetika metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.
- MeSH
- "flap" endonukleasy genetika metabolismus terapeutické užití MeSH
- enzymy opravy DNA genetika MeSH
- exodeoxyribonukleasy genetika MeSH
- glykosidhydrolasy genetika metabolismus MeSH
- lidé MeSH
- nádorový supresorový protein p53 * genetika metabolismus MeSH
- nádory * farmakoterapie genetika MeSH
- oprava DNA MeSH
- PARP inhibitory farmakologie MeSH
- poškození DNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- exodeoxyribonukleasy genetika metabolismus MeSH
- fosfoproteiny genetika metabolismus MeSH
- lidé MeSH
- mutace * MeSH
- myši MeSH
- rekombinasa Rad51 biosyntéza genetika metabolismus MeSH
- replikace DNA * MeSH
- transfekce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
INTRODUCTION AND AIM: Infections caused by herpes simplex viruses (HSV) are frequent in the human population. Because of the widespread use of long-term treatment or prophylaxis by anti-herpetic antivirals in various specific medical contexts (immunosuppression, recurrent infections), the level of antiviral resistance is increasing. According to previous studies, there is a low resistance level in immunocompetent populations but a relatively high level in populations with immunodeficiency. However, there has been no study from the Czech Republic. This study presents results of a single-centre retrospective study from the Czech Republic. MATERIALS AND METHODS: Deep frozen DNA from patients with suspected clinical antiviral failure over a long time period (2009-2016) - a total of 15 isolates of HSV1 and seven of HSV2 - were examined for the presence of mutations associated with antiviral resistance. Sequence analysis was performed using an ABI PRISM 3500xL Genetic Analyzer (Applied Biosystems®). RESULTS: There were no mutations associated with resistance to antivirals inside the UL23 gene in HSV1 isolates. However, resistant mutation D672N (nucleotide change G2014A) was found inside the UL30 gene in seven of the isolates. One mutation associated with resistance to acyclovir (M183stop) was found inside the UL23 gene in one HSV2 isolate. Resistant mutation E678G (nucleotide change A2033G) was identified inside the UL30 gene in six of the HSV2 isolates. CONCLUSIONS: This study confirmed the presence of resistance mutations within the Czech population, but it will be necessary to examine a higher number of isolates for further conclusions.
- MeSH
- acyklovir farmakologie MeSH
- antivirové látky farmakologie MeSH
- DNA-dependentní DNA-polymerasy genetika MeSH
- exodeoxyribonukleasy genetika MeSH
- herpes simplex farmakoterapie virologie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- mutace MeSH
- neúspěšná terapie MeSH
- retrospektivní studie MeSH
- Simplexvirus účinky léků genetika MeSH
- virová léková rezistence genetika MeSH
- virové proteiny genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51(T131P) mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.
- MeSH
- časové faktory MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- DNA-helikasy genetika metabolismus MeSH
- DNA-polymerasa I metabolismus MeSH
- DNA-polymerasa III metabolismus MeSH
- DNA biosyntéza genetika MeSH
- endodeoxyribonukleasy genetika metabolismus MeSH
- exodeoxyribonukleasy genetika metabolismus MeSH
- lidé MeSH
- mutace MeSH
- nestabilita genomu MeSH
- protein BRCA2 genetika metabolismus MeSH
- proteiny Xenopus genetika metabolismus MeSH
- rekombinasa Rad51 genetika metabolismus MeSH
- replikace DNA * MeSH
- replikační počátek MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Xenopus laevis genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Protein modifications regulate both DNA repair levels and pathway choice. How each modification achieves regulatory effects and how different modifications collaborate with each other are important questions to be answered. Here, we show that sumoylation regulates double-strand break repair partly by modifying the end resection factor Sae2. This modification is conserved from yeast to humans, and is induced by DNA damage. We mapped the sumoylation site of Sae2 to a single lysine in its self-association domain. Abolishing Sae2 sumoylation by mutating this lysine to arginine impaired Sae2 function in the processing and repair of multiple types of DNA breaks. We found that Sae2 sumoylation occurs independently of its phosphorylation, and the two modifications act in synergy to increase soluble forms of Sae2. We also provide evidence that sumoylation of the Sae2-binding nuclease, the Mre11-Rad50-Xrs2 complex, further increases end resection. These findings reveal a novel role for sumoylation in DNA repair by regulating the solubility of an end resection factor. They also show that collaboration between different modifications and among multiple substrates leads to a stronger biological effect.
- MeSH
- DNA vazebné proteiny genetika MeSH
- dvouřetězcové zlomy DNA MeSH
- endodeoxyribonukleasy genetika MeSH
- endonukleasy genetika MeSH
- exodeoxyribonukleasy genetika MeSH
- fosforylace MeSH
- lidé MeSH
- oprava DNA spojením konců genetika MeSH
- oprava DNA genetika MeSH
- poškození DNA genetika MeSH
- rozpustnost MeSH
- Saccharomyces cerevisiae - proteiny genetika MeSH
- Saccharomyces cerevisiae MeSH
- sumoylace genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural 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.
- 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
- 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
Various processes of bacteriophage lambda development in Escherichia coli cells bearing either the whole lambda exo-xis region (with truncated, thus nonfunctional, exo and xis genes) or particular genes from this region were investigated. The presence of either the exo-xis region or the ea8.5 gene on a plasmid resulted in formation of fuzzy plaques by infecting phage. Both efficiency of plating and efficiency of lysogenization were decreased in such hosts. On the other hand, neither the efficiency of adsorption nor intracellular lytic development of the infecting phage (measured in one-step-growth experiments) was affected while significantly more host cells survived the infection, when containing the exo-xis region. Although no effects of the exo-xis region on the activity of the p (L) promoter was detected, this region contributed to a decreased transcription from the cII-stimulated promoters p (I), p (aQ) and p (E). These results, together with the results of measurement of efficiency of plating of phages bearing mutations in cI, cII and cIII genes on hosts containing the exo-xis region, strongly suggest that genes from this region (especially ea8.5) are involved in the regulation of bacteriophage lambda development at the stage of the lysis-vs.-lysogenization decision.
- MeSH
- bakteriofág lambda fyziologie genetika růst a vývoj MeSH
- bakteriolýza MeSH
- DNA-nukleotidyltransferasy genetika chemie MeSH
- Escherichia coli virologie MeSH
- exodeoxyribonukleasy genetika chemie MeSH
- lyzogenie MeSH
- mutace MeSH
- plakové testy MeSH
- regulace exprese virových genů MeSH
- virové proteiny genetika chemie metabolismus MeSH
