Project description:Deletion mutants of the yeast MRX complex (Rad50, Mre11 and Xrs2) were studied for gene expression levels. Significant difference in expression levels was observed in genes pertaining to DNA damage, cell cycle etc. as compared to the BY4741 wild type.
Project description:Mre11, together with Rad50 and Xrs2/NBS, plays pivotal roles in homologous recombination, repair of DNA double strand breaks (DSBs), activation of damage-induced checkpoint, and telomere maintenance. Using DNA microarray assays to analyze yeast mutants (mre11delta, rad50delta, and spo11Y135F) defective for meiotic DSB formation, we demonstrate that the absence of Mre11 in yeast causes specific effects on regulation of a class of meiotic genes for spore development. The transcriptional deficiency was not observed in other DSB mutants such as rad50delta and spo11Y135F, suggesting the transcriptional defect in mre11delta is due to neither lack of meiotic DSB formation, nor disintegrity of Mre11-Rad50-Xrs2 complex.These defects were confirmed by northern and lacZ reporter gene assays. Experiment Overall Design: Gene expression data from wild type, mre11delta, rad50delta, and spo11Y135F cells in premeiosis (meiosis 0 hr) and prophase (meiosis 4 hr). Affymetrix GeneChip YG-S98 was used. All strains used were SK1 background diploid cells.
Project description:Deletion mutants of the yeast MRX complex (Rad50, Mre11 and Xrs2) were studied for gene expression levels. Significant difference in expression levels was observed in genes pertaining to DNA damage, cell cycle etc. as compared to the BY4741 wild type. Organism : Saccharomyces cerevisiae , Custom Saccharomyces cerevisiae 8x15k Gene expresssion Microarray (AMADID: 016333) designed by Genotypic Technology Private Limited
Project description:he human MRE11/RAD50/NBS1 (MRN) complex plays a crucial role in sensing and repairing DNA DSB. MRE11 possesses 3’-5’ exonuclease and endonuclease activity and forms the core of the multifunctional MRN complex. We previously identified a C-terminally truncated form of MRE11 (TR-MRE11) associated with post-translational MRE11 degradation. Here we identified the approximate cleavage site of TR-MRE11 at 559-580 amino acids, its DNA damage repair function and the factors regulating TR-MRE11 accumulation. The nuclease enzymatic activity of TR-MRE11 was dramatically reduced, associated with a lack of DNA binding efficiency, whilst TR-MRE11 still interacted efficiently with RAD50 and NBS1. Lack of the MRE11 C-terminal resulted in deficient HR repair and increased cellular radiosensitivity. Knockdown of SprT-like N-terminal domain (SPRTN), an essential metalloprotease for DNA-protein crosslink repair, resulted in failure of MRE11 cleavage, with TR-MRE11 protein levels being positively correlated with SPRTN protein expression. The presence of this DNA repair-defective C-terminal truncation could explain the finding of high MRE11 expression, by immunohistochemistry using an antibody against MRE11 prior to the C-terminal, being associated with survival following radical radiotherapy in cancer patients. Ultimately, understanding the functional differences between intact and repair-defective MRE11 may lead to improvements in patient outcomes through a more informed choice of treatment.
Project description:Rad50 is a component of the conserved MRE11-RAD50-NBS1 (MRN) complex, which functions in genome stability and the cell’s ability to deal with stalled DNA replication forks. We identified Rad50 as a factor important for R-loop tolerance and thus mapped DNA:RNA hybrids in Rad50KO cells and compare them to previously reported wild-type and Sgs1KO profiles.
Project description:Mre11, together with Rad50 and Xrs2/NBS, plays pivotal roles in homologous recombination, repair of DNA double strand breaks (DSBs), activation of damage-induced checkpoint, and telomere maintenance. Using DNA microarray assays to analyze yeast mutants (mre11delta, rad50delta, and spo11Y135F) defective for meiotic DSB formation, we demonstrate that the absence of Mre11 in yeast causes specific effects on regulation of a class of meiotic genes for spore development. The transcriptional deficiency was not observed in other DSB mutants such as rad50delta and spo11Y135F, suggesting the transcriptional defect in mre11delta is due to neither lack of meiotic DSB formation, nor disintegrity of Mre11-Rad50-Xrs2 complex.These defects were confirmed by northern and lacZ reporter gene assays. Keywords: mutant vs. wildtype strains
Project description:Programed DNA double-strand breaks (DSBs) catalyzed by the topoisomerase II-like enzymes, SPO11 and TOPVIBL, initiate meiotic recombination. Following DSB formation, the MRE11-RAD50-NBS1/Xrs2 (MRN/X) complex, along with EXO1 and DNA2, cleave the SPO11-DNA to generate 3′ single-stranded DNA (ssDNA) ends, which are prerequisite for meiotic DSB repair. In both yeast and mammals, MRE11 exhibits endonucleolytic cleavage of the 5′ terminated DNA strand in the vicinity of the DSBs and exonucleolytic resection from 3′ to 5′ towards the DSB ends. RAD50 is a structure maintenance of chromosome (SMC) related protein that contains one ATPase domain at its N- and C- terminal ends, respectively, Zn hook, and anti-parallel coiled coils. RAD50 plays a crucial role in facilitating the MRE11 nuclease activity on DSBs by ATP binding and hydrolysis. However, the in vivo function of Rad50 in mammalian germ cells, particularly its in vivo role in the resection of meiotic DSB ends at the molecular level remains elusive. Here, we performed END-seq with synchronized zygotene spermatocytes from control and germ cell specific Rad50 mutant (Rad50-sKO) mice. We find that the number of formed DSB in the mutant spermatocytes was reduced compared to control spermatocytes (6 636 DSBs in Rad50-sKO spermatocytes vs 8 168 in control spermatocytes) and abnormal DSB end resection occurred in mutant spermatocytes (DSB end resection length: 1 279 nts in Rad50-sKO spermatocytes vs 1 923 nts in control spermatocytes). Thus, RAD50 is essential for DSB formation and end resection during mammalian meiosis.