Influence of chromatin modificationand nuclease activities in controlling the cellular response to DNA damage

The DNA damage checkpoint coordinates cell-cycle progression, DNA repair, replication and recombination in response to DNA damage. Defects in this surveillance mechanism lead to genomic instability, cancer susceptibility, ageing and several human pathologies. It is generally accepted that the DNA damage response is triggered by the accumulation of long single-stranded DNA (ssDNA) regions. While the basics of the mechanisms producing the ssDNA tails after double strand breaks (DSBs) have been investigated, it is not clear how these long ssDNA regions are generated following UV treatment. We showed that nucleotide excision repair (NER), the sole pathway able to remove UV-induced lesions and a major defence against the carcinogenic effects of UV light, is required for triggering the DNA damage response after UV radiation, both in yeast and in human cells. However, NER is very fast and efficient and produces only short (25-30 nt) ssDNA gaps. By using Saccharomyces cerevisiae as model system, we obtained evidence for a role of Exo1 and other yet unidentified nucleases in the checkpoint after UV. The model arising from these studies is that most UV photoproducts are rapidly repaired by NER, but some of the lesions are further processed by Exo1 generating long ssDNA regions that activate the checkpoint. In order to test this hypothesis we are planning to use a variation of the DNA combing technique to directly visualize these regions on the yeast chromosomes and to establish whether the Exo1 processed lesions are randomly distributed in the genome or localized at specific loci, as preliminary experiments suggest. The findings obtained in the yeast system will be extended to human cells, investigating the role of the two hEXO1 splice variants in the UV response by using a combination of approaches. The DNA damage response is dramatically influenced by chromatin. Histone modifications and chromatin remodeling factors play important roles in the activation of the cascade and in the recruitment of checkpoint/repair proteins to damaged chromosomes. This aspect can be studied exploiting a model DSB, which is inducible and site-specific, allowing precise monitoring of both DSB processing and loading of specific proteins on the damaged DNA. In yeast, the cell cycle position of the damaged cell strongly influences how DSBs are treated. DSB processing is inhibited in G1; this inhibition is relieved upon activation of Cdk1/Clb kinase. Several factors are involved in generating ssDNA tails from a DSB, namely Mre11-Rad50-Xrs2 , Sae2 and Exo1, even though the exact mechanism is still not clear. The ssDNA filaments are rapidly coated by RPA, activating Mec1 checkpoint kinase, and are channeled into the recombinational repair pathway. We have found that checkpoint activation after a DSB requires histone H3-K79 methylation, providing a binding site for the Rad9 checkpoint factor on chromatin. Preliminary evidence suggests that methylation-dependent binding of Rad9 also has a negative effect on Mec1 activation through the inhibition of the DSB processing activity. We propose to study the role played by chromatin modifications on the mechanisms controlling DSB processing and signaling. We will define how methylation of H3 inhibits resection by analyzing th