Romain Chayot - Academia.edu (original) (raw)
Papers by Romain Chayot
Stem Cell Research, 2014
The loss of genome integrity in adult stem cells results in accelerated tissue aging and possibly... more The loss of genome integrity in adult stem cells results in accelerated tissue aging and possibly cancerogenesis. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fibre exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs.
Proceedings of the National Academy of Sciences, 2010
Bridging broken DNA ends via nonhomologous end-joining (NHEJ) contributes to the evolution and st... more Bridging broken DNA ends via nonhomologous end-joining (NHEJ) contributes to the evolution and stability of eukaryote genomes. Although some bacteria possess a simplified NHEJ mechanism, the human commensal Escherichia coli is thought to rely exclusively on homology-directed mechanisms to repair DNA double-strand breaks (DSBs). We show here that laboratory and pathogenic E. coli strains possess a distinct end-joining activity that repairs DSBs and generates genome rearrangements. This mechanism, named alternative end-joining (A-EJ), does not rely on the key NHEJ proteins Ku and Ligase-D which are absent in E. coli. Differently from classical NHEJ, A-EJ is characterized by extensive end-resection largely due to RecBCD, by overwhelming usage of microhomology and extremely rare DNA synthesis. We also show that A-EJ is dependent on the essential Ligase-A and independent on Ligase-B. Importantly, mutagenic repair requires a functional Ligase-A. Although generally mutagenic, accurate A-EJ also occurs and is frequent in some pathogenic bacteria. Furthermore, we show the acquisition of an antibioticresistance gene via A-EJ, refuting the notion that bacteria gain exogenous sequences only by recombination-dependent mechanisms. This finding demonstrates that E. coli can integrate unrelated, nonhomologous exogenous sequences by end-joining and it provides an alternative strategy for horizontal gene transfer in the bacterial genome. Thus, A-EJ contributes to bacterial genome evolution and adaptation to environmental challenges. Interestingly, the key features of A-EJ also appear in A-NHEJ, an alternative end-joining mechanism implicated in chromosomal translocations associated with human malignancies, and we propose that this mutagenic repair might have originated in bacteria.
DNA Repair, 2010
The specialised DNA polymerase (pol ) affects a sub-class of immunoglobulin genes rearrangements ... more The specialised DNA polymerase (pol ) affects a sub-class of immunoglobulin genes rearrangements and haematopoietic development in vivo. These effects appear linked to double-strand breaks (DSBs) repair, but it is still unclear how and to what extent pol intervenes in this process. Using high-resolution quantitative imaging of DNA damage in irradiated wild-type and pol −/− mouse embryonic fibroblasts (MEFs) we show that lack of pol results in delayed DSB repair kinetics and in persistent DNA damage. DNA damage triggers cellular senescence, and this response is thought to suppress cancer. Independent investigations either report or not a proliferative decline for MEFs lacking pol . Here we show pronounced senescence in pol −/− MEFs, associated with high levels of the tumor-suppressor p16 INK4A and the DNA damage response kinase CHK2. Importantly, cellular senescence is induced by culture stress and exacerbated by low doses of irradiation in pol −/− MEFs. We also found that low doses of irradiation provoke delayed immortalisation in MEFs lacking pol . Pol −/− MEFs thus exhibit a robust anti-proliferative defence in response to irreparable DNA damage. These findings indicate that sub-optimal DSB repair, due to the absence of an auxiliary DNA damage repair factor, can impact on cell fitness and thereby on cell fate.
DNA Repair, 2012
The specialized DNA polymerase (pol ) intervenes in the repair mechanism non-homologous endjoinin... more The specialized DNA polymerase (pol ) intervenes in the repair mechanism non-homologous endjoining (NHEJ) as an end-processing factor but its role has not been fully elucidated. Pol has been shown to participate in DNA synthesis at junctions in vitro, including on unpaired substrates, and to promote annealing. However, the phenotypes observed in vivo poorly recapitulate the functions of pol reported in vitro. We analysed the repair of DNA double-strand breaks (DSBs) in a cellular context using improved NHEJ substrates. These substrates do not replicate in mammalian cells, thereby result in clonal repair events, which allows the measure of the efficiency of repair. We validated this paradigm by comparing the repair of NHEJ substrates to the repair reported for chromosome DSBs in mouse cells. Molecular analysis and, in most cases sequencing of more than 1500 repair events on a variety of NHEJ substrates in wild type and pol −/− mouse embryonic fibroblasts shows that, unexpectedly, the absence of pol decreases the efficiency of joining of all types of DSBs, including those that do not undergo end-processing. Importantly, by reducing the efficiency of accurate events, lack of pol also affects the overall fidelity of the repair process. We also show that, although pol does not help protect DNA ends from resection, the efficiency of repair of resected ends is reduced in the absence of pol . Interestingly, the DNA synthesis activity of pol , including on non-aligned substrates, appears negligible at least in a cellular context. Our data point to a critical role for pol as a global repair player that increases the efficiency and the fidelity of NHEJ.
Stem Cell Research, 2014
The loss of genome integrity in adult stem cells results in accelerated tissue aging and possibly... more The loss of genome integrity in adult stem cells results in accelerated tissue aging and possibly cancerogenesis. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fibre exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs.
Proceedings of the National Academy of Sciences, 2010
Bridging broken DNA ends via nonhomologous end-joining (NHEJ) contributes to the evolution and st... more Bridging broken DNA ends via nonhomologous end-joining (NHEJ) contributes to the evolution and stability of eukaryote genomes. Although some bacteria possess a simplified NHEJ mechanism, the human commensal Escherichia coli is thought to rely exclusively on homology-directed mechanisms to repair DNA double-strand breaks (DSBs). We show here that laboratory and pathogenic E. coli strains possess a distinct end-joining activity that repairs DSBs and generates genome rearrangements. This mechanism, named alternative end-joining (A-EJ), does not rely on the key NHEJ proteins Ku and Ligase-D which are absent in E. coli. Differently from classical NHEJ, A-EJ is characterized by extensive end-resection largely due to RecBCD, by overwhelming usage of microhomology and extremely rare DNA synthesis. We also show that A-EJ is dependent on the essential Ligase-A and independent on Ligase-B. Importantly, mutagenic repair requires a functional Ligase-A. Although generally mutagenic, accurate A-EJ also occurs and is frequent in some pathogenic bacteria. Furthermore, we show the acquisition of an antibioticresistance gene via A-EJ, refuting the notion that bacteria gain exogenous sequences only by recombination-dependent mechanisms. This finding demonstrates that E. coli can integrate unrelated, nonhomologous exogenous sequences by end-joining and it provides an alternative strategy for horizontal gene transfer in the bacterial genome. Thus, A-EJ contributes to bacterial genome evolution and adaptation to environmental challenges. Interestingly, the key features of A-EJ also appear in A-NHEJ, an alternative end-joining mechanism implicated in chromosomal translocations associated with human malignancies, and we propose that this mutagenic repair might have originated in bacteria.
DNA Repair, 2010
The specialised DNA polymerase (pol ) affects a sub-class of immunoglobulin genes rearrangements ... more The specialised DNA polymerase (pol ) affects a sub-class of immunoglobulin genes rearrangements and haematopoietic development in vivo. These effects appear linked to double-strand breaks (DSBs) repair, but it is still unclear how and to what extent pol intervenes in this process. Using high-resolution quantitative imaging of DNA damage in irradiated wild-type and pol −/− mouse embryonic fibroblasts (MEFs) we show that lack of pol results in delayed DSB repair kinetics and in persistent DNA damage. DNA damage triggers cellular senescence, and this response is thought to suppress cancer. Independent investigations either report or not a proliferative decline for MEFs lacking pol . Here we show pronounced senescence in pol −/− MEFs, associated with high levels of the tumor-suppressor p16 INK4A and the DNA damage response kinase CHK2. Importantly, cellular senescence is induced by culture stress and exacerbated by low doses of irradiation in pol −/− MEFs. We also found that low doses of irradiation provoke delayed immortalisation in MEFs lacking pol . Pol −/− MEFs thus exhibit a robust anti-proliferative defence in response to irreparable DNA damage. These findings indicate that sub-optimal DSB repair, due to the absence of an auxiliary DNA damage repair factor, can impact on cell fitness and thereby on cell fate.
DNA Repair, 2012
The specialized DNA polymerase (pol ) intervenes in the repair mechanism non-homologous endjoinin... more The specialized DNA polymerase (pol ) intervenes in the repair mechanism non-homologous endjoining (NHEJ) as an end-processing factor but its role has not been fully elucidated. Pol has been shown to participate in DNA synthesis at junctions in vitro, including on unpaired substrates, and to promote annealing. However, the phenotypes observed in vivo poorly recapitulate the functions of pol reported in vitro. We analysed the repair of DNA double-strand breaks (DSBs) in a cellular context using improved NHEJ substrates. These substrates do not replicate in mammalian cells, thereby result in clonal repair events, which allows the measure of the efficiency of repair. We validated this paradigm by comparing the repair of NHEJ substrates to the repair reported for chromosome DSBs in mouse cells. Molecular analysis and, in most cases sequencing of more than 1500 repair events on a variety of NHEJ substrates in wild type and pol −/− mouse embryonic fibroblasts shows that, unexpectedly, the absence of pol decreases the efficiency of joining of all types of DSBs, including those that do not undergo end-processing. Importantly, by reducing the efficiency of accurate events, lack of pol also affects the overall fidelity of the repair process. We also show that, although pol does not help protect DNA ends from resection, the efficiency of repair of resected ends is reduced in the absence of pol . Interestingly, the DNA synthesis activity of pol , including on non-aligned substrates, appears negligible at least in a cellular context. Our data point to a critical role for pol as a global repair player that increases the efficiency and the fidelity of NHEJ.