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Mechanisms coordinating chromosome replication with transcription

Final Report Summary - REPTRA (Mechanisms coordinating chromosome replication with transcription)

Chromosome instability is hallmark of cancer cells and cellular aging. The DNA damage response (DDR) is mediated by Mec1 and Tel1 in budding yeast, and by two downstream kinases, Rad53 and Chk1. Mec1 and Tel1 respond to different types of DNA lesions, to which they are recruited through specific factors. Mec1 is primarily activated in cells undergoing replication stress by DDC2 and RFA−coated ssDNA). However, Mec1 is also activated by DSBs through a mechanism requiring Tel1. While Mec1 role in response to DNA damage has been extensively studied, its essential function in response to endogenous stress is less clear. As described in my proposal, the host laboratory has identified the suppressors of rad53 HU sensitivity. Of these, mutants in genes encoding THO/TREX complex involved in mRNP biogenesis were of particular interest due to the importance of replication fork stability at transcribing genes. The overarching aim of the research proposal is to understand the mechanisms coordinating replication across transcribed units using budding yeast/ mammalian cell lines as a model organism. To address the scientific objectives, we divided the proposal in two tasks. A significant progress was made towards the objective in the host lab to understand the mechanism of mec1 and rad53-mediated control of replication fork stability at the transcribing regions and in fact some of the findings have already been published (Bermejo et. Al., 2011, Cell). Various budding yeast strains required for the study were constructed and chip on chip for subunits of THO/TREX complex were performed in wt and rad53 strains undergoing replication stress. Accumulation of ssDNA in rad53 cells was also studied using chip on chip and the clusters were compared to genomic loci enriched in HPR1 subunit of THO/TREX complex. The finding that Hpr1 recruitment was increased in checkpoint defective strains undergoing replication stress, led us to examine genomic loci enriched in RNA-DNA hybrids using chip on chip in different THO/TREX subunit mutants. No significant impact on the fragility of the chromosomes was observed in mutants exhibiting dysfunctional THO/TREX complex with increased incidence of RNA:DNA hybrids accumulation. In addition to the continuation with the proposed objectives, we started characterizing intra-cellular localization of Mec1 and subunits of THO/TREX complex and in course of time moved to mammalian cell lines to study the additional new aims using advanced imaging techniques. A significant progress was achieved during second year and the findings have been submitted to Cell Journal for publication (Kumar & Foiani et al.,). We found that human ATR, ATRIP and Chk1 associate with the nuclear envelope during S phase and prophase. Osmotic stress relocalizes ATR to the nuclear membrane throughout the cell cycle; mechanical stimulation of the nuclear membranes accumulates ATR at the nuclear envelope. The ATR-mediated mechanical response occurs within the range of physiological forces, recovers rapidly, and does not require RPA or DNA damage. ATR-defective cells exhibit aberrant chromatin condensation and nuclear envelope breakdown.We propose that mechanical forces owing to chromosome dynamics or mechanical stress on nuclear membranes activate ATR that counteracts DNA topological stress and cytoskeleton forces at perinuclear chromatin by modulating nuclear envelope plasticity and chromatin association. Thus, ATR is part of an integrated mechanical response coupling nuclear envelope-induced signaling with epigenetic changes within the nucleus. My second task was to characterize the checkpoint-mediated epigenetic control of S phase transcription. To achieve the objectives, I have constructed different budding yeast strains during the period and has performed chip on chip analysis for Topoisomerase 2 (Top2) localization. Top2 genomic localization was examined in S-phase of the cells in WT and checkpoint defective (Rad53-K227A) strains undergoing replication stress. No significant differences were observed suggesting that Top2 localization at intergenic regions is independent of DNA checkpoint proteins.