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    • Given the recent discovery of regions of single stranded

    2022-12-01

    Given the recent discovery of regions of single-stranded DNA in the nuclei of hypoxic cells, the role for ATR in the response to hypoxic stress seems clearer . Zou and Elledge demonstrated that both Ddc 2 (the homolog of ATRIP) and ATRIP have a lower affinity for double-strand breaks than they do for RPA–ssDNA complexes and concluded that ssDNA coupled with RPA recruits ATR/ATRIP. Several critical unanswered questions remain. In response to hypoxia, does ATR respond purely to the replication arrest rather than replication arrest-induced DNA damage? Why are molecules such as Rad 17, Nbs 1 and histone H2AX phosphorylated in hypoxic AZ 10417808 australia when there is no evidence of DNA breaks? There are several possibilities; hypoxia-induced stalled replication forks do become damaged, but are repaired quickly and efficiently in the presence of ATR. The most likely form of damage at a replication fork is however a double-strand break, the half-life of which is approximately 2h making it unlikely that they would not be detected by comet assay unless they are so few in number to be below the sensitivity of this assay. We would predict that given the rapid phosphorylation of Nbs 1 in response to hypoxia, the MRN complex, consisting of Mre 11, Rad 50 and Nbs 1, would have a role to play in the cellular response to extreme hypoxia. It was recently proposed that random DNA lesions could be converted to structures more amenable to both repair and the recruitment of signal transducers by the MRN complex, therefore, raising the possibility that hypoxia-induced stalled replication forks are somehow modified by the MRN complex . Studies of the MRN orthologs in both bacteria and bacteriophage T4 have shown that the complex is required for both processing of DNA structures that can arise at replication forks and recombination-dependent replication and re-start of stalled replication forks , , . Nbs 1 has been shown to have a role in homologous recombination, a process which has also been linked to replication re-start . Mre 11 has also recently been found to have a role to play in recruiting ATM and probably ATR to the sites of either damage or stalled replication forks therefore placing the MRN complex both up and downstream of ATM/ATR in the signaling pathway. This finding does in some part explain the similarities in A-T and A-TLD which arises in patients with hypomorphic Mre11 mutations . At present the ATR signaling pathway in A-TLD cell lines has not been investigated. Recent reports from both yeast and mammalian cells indicate that ATR has a role to play in preserving the integrity of stalled replication forks and that in the absence of functional ATR forks become damaged . In accordance with these reports, when ATR is inhibited during hypoxia treatment cells accumulate DNA damage that is specific to S-phase cells (Hammond and Giaccia, unpublished data). This finding strongly concurs with published results, and implies a definite role for ATR in the maintenance of replication forks during both stress and normal cell division , . Rad 53, the functional homolog of mammalian Chk 1, was also demonstrated to prevent replication fork collapse which raises the possibility that Chk 1 may do the same in response to hypoxia. These recent findings from both yeast and mammalian biology lead to the question, why, in contrast to both ATM and DNA-PK, is ATR an absolutely essential gene? Further insight into the absolute requirement for ATR may come from studying other molecules in these signaling cascades which are also embryonic lethal or only exist in a hypomorphic state, for example, Rad 17, Chk 1, Nbs 1 and Mre11. Stalled replication occurs during normal cell division at fragile sites and also during physiologically relevant stresses such as hypoxia and UV exposure . During hypoxia the principle role of ATR may well be to protect cells from acquiring DNA damage during the most sensitive phase of the cell cycle, S-phase, by inducing repair pathways, cell-cycle arrest or apoptosis. Interestingly both ATR and Chk 1 have recently been shown to inhibit replicon initiation following UVC-induced DNA damage . This finding may have implications for the mechanism of hypoxia-mediated replication arrest; perhaps ATR and the Chk 1 downstream pathway have a role to play?