The DNA damage checkpoint is organized as a signal transduction cascade where the Mec1-Ddc2 kinase complex and the Mec3-Ddc1-Rad17 (PCNA-like) complex are involved in the earlier steps of checkpoint signal generation and transmission, while Rad53 is the major downstream effector kinase. It has been shown that a DNA double strand break is processed to generate a long 3’OH single-stranded DNA tails covered by RPA, and this particular structure seems to be necessary for recruiting checkpoint complexes near the lesion. The details of the molecular mechanisms required for generation of the checkpoint signal after UV-induced DNA damage still need to be defined. We have recently shown that NER proteins and checkpoint factors physically interact and that recognition and processing of UV lesions by NER factors are required for recruitment of checkpoint complexes onto damaged chromosomes. In a two-hybrid screen, using the Ddc1 subunit of the PCNA-like complex as bait, we identified Exo1 as a strong Ddc1 interactor. Exo1 is a 5’-3’ exonuclease with multiple roles in DNA repair, recombination, replication and telomeres integrity. Further analysis has shown that Exo1 is involved in the early steps of the checkpoint activation in the presence of UV induced DNA damage. Infact, its exonuclease activity is required to create the signal for the recruitment of Mec1-Ddc2 and for the early activation of the checkpoint following UV damage. We showed that epigenetic modifications of histones, and specifically H2B-K123 ubiquitination by Rad6-Bre1, and H3-K79 methylation by Dot1, are necessary for the proper G1 checkpoint response to a variety of genotoxic agents. In particular, these modifications seem to be needed for the recruitment and phosphorylation of Rad9. In fact cells carrying mutations within the Tudor domain of Rad9 display the same defects as H3-K79A mutants. Intriguingly, this mechanism seems to be essential only during G1 and S phase, while the existence of a redundant mechanism for Rad9 recruitment in G2 cells is likely. In fact, loss of H3-K79 methylation affects G2 checkpoint activation only partially. We are actively searching for other factors that may be involved in this redundant pathway. Recent results suggest that these epigenetic modifications of chromatin also contribute to regulating the extent of Exo1-dependent processing of damaged DNA.
|Titolo:||Exo1 is required for an early step of checkpoint activation in response to UV-irradiation in yeast cells and is regulated by epigenetic modifications of chromatin|
|Data di pubblicazione:||2006|
|Settore Scientifico Disciplinare:||Settore BIO/11 - Biologia Molecolare|
Settore BIO/18 - Genetica
|Citazione:||Exo1 is required for an early step of checkpoint activation in response to UV-irradiation in yeast cells and is regulated by epigenetic modifications of chromatin / M. Giannattasio, F. Lazzaro, F. Goffredo, M. Granata, F. Puddu, C. Visintin, P. Plevani, M. Muzi-Falconi. ((Intervento presentato al convegno FASEB Yeast chromosome structure. replication & segregation tenutosi a Indian Wells, USA nel 2006.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|