[“Molecular Evolutionary Genetics Analysis

Chromatinremodeling complexes use the energy from ATP hydrolysis to induce these changes, leading to the mobilization and repositioning of histone octamers in cis. For instance, the yeast SWR complex associates with the HA variant histone HA.Indeed, several proteins do recognize gHAX, or the yeast equivalent, gHA, in vitro.Several mammalian DDR proteins contain forkheadassociated domains domains that are able to interact with phosphorylated threonine or serine residues. Phosphopeptidebinding studies rst revealed MDC as a ligand of gHAX in whole human cell extracts. The interaction was later conrmed in a peptidescreen using the puried MDC tandem BRCT domain and the relevance of this interaction was conrmed by showing that mutations in either the HAX phosphoacceptor site or in the conserved residues of BRCT domain impair MDC accumulation in IRIF.Thus, MDC seemed to be an early regulator of the DDR, contributing to both the recruitment and the retention of other DDR factors.Database searches failed to reveal homologues of MDC in plants or lower eukaryotes such as yeast, indicating that the function of MDC in the DDR is unique to mammals.Their functional domains and posttranslational modifications are indicated.CK constitutively phosphorylates residues within the SDT region of MDC, which is then bound by the FHA domain of NBS.RNF contains a RING finger domain that regulates ubiquitylation of gHAX and HA.RNF binds to ubiquitylated histones and, like RNF, contains a RING finger domain that promotes the formation of ubiquitin conjugates.The kinetics of recruitment and accumulation of DDR factors at sites of damage were elucidated by livecell imaging studies.There is substantial variation in the dynamic behavior of DDR proteins at sites of DNA damage.For instance, NBS and MDC, which are the rst factors recruited to the DSB, bind within a few seconds of damage induction. BP and BRCA are also rapidly recruited, although their appearance is signicantly slower than that of NBS and MDC.Importantly, IRIF formation by NBS, BP and BRCA failed in strains lacking MDC. This complex binds and activates the checkpoint kinase ATM. These observations suggest a model for the formation and spread of gHAX, and for the accumulation and retention of DDR proteins at sites of damage.Initially, the free ends of DNA at a DSB are recognized by MRN, which mediates an immediate recruitment of ATM.MDC is then recruited and bound to gHAX, which promotes purchase Abamectin further MRNATM recruitment and a subsequent extension of HAX phosphorylation to anking nucleosomes. The repetition of these latter events can create a positive feedback loop that extends the gHAX domain beyond the site of damage. The phosphorylation of the SDT repeats of MDC by CK seems to have an important role for the recruitment, accumulation and retention of the MRN complex and, as such, can contribute to gHAX propagation as well. These results support a model in which MRN initiates the formation and spread of gHAX.ATM, which is bound to MDC via MRN, phosphorylates MDC to recruit an RNFUBC complex that seems to regulate ubiquitylation of histone HAX and HAX.RNF binds to these ubiquitylated histones and promotes the formation of ubiquitin conjugates.

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