Research

RecQ helicases and Genomic Integrity One major question in cancer genetics is to what extent chromosomal instability is the beginning of tumorigenesis. RecQ helicases have been called caretakers of the genome and it is well documented that the loss of RecQ function leads to the breakdown in the maintenance of genome integrity. RecQ helicases likely maintain genomic stability by functioning at the interface between DNA replication and DNA repair and defects in these helicases are known to lead to an increase in cancer predisposition. In humans there are five known RecQ helicases, three of which are associated with genetic diseases. The BLM, WRN and RECQ4 genes are mutated in Bloom's syndrome (BS), Werner's syndrome (WS), and Rothmund-Thomson syndrome (RTS) respectively. Both WS and BS cells show a high frequency of chromosome rearrangements, sister chromatid exchange, recombination and the accumulation of abnormal DNA replication intermediates. Additionally WS is characterized by the premature appearance of aging phenotypes in young adults and an elevated incidence of rare sarcomas. We are elucidating the function of RecQ at the molecular level using Saccharomyces cerevisiae (budding yeast) as a model system where there is one RecQ helicase called Sgs1. It is quite evident that mechanisms that preserve genome stability in yeast are indeed the same as those which go awry in many mammalian cancers. We are focusing on the relationship between Sgs1 and the MRX complex and determining if RecQ helicases preserve fragile site stability in slow replicating regions of the genome.

Genomic Fragile Sites Conditions that partially inhibit replication induce breaks in the DNA more frequently at certain chromosomal locations termed fragile sites. These sites form breaks on metaphase chromosomes and are deleted and rearranged in many tumors. Still, the mechanisms leading to damage at genomic fragile sites remains elusive, and to date there is no systematic study linking fragile sites to gross chromosomal rearrangements (GCRs) at stalled replication forks at particular genomic loci. Fragile sites are normally stable in cultured human cells. The checkpoint kinase ATR (ScMec1), but not ATM (ScTel1) is critical for maintenance of fragile site stability and following induction with replication inhibitors these sites are "hot spots" for chromosomal rearrangements. Astonishingly, breaks at just 20 fragile sites represent over 80% of all lesions observed in lymphocytes following treatment with low doses of the replication inhibitor aphidicolin. Studies in fission yeast have shown that recombination events take place at the site of collapsed forks and that this event is a precursor to GCRs. These events increase dramatically in the absence of RecQ helicase. We are investigating the role of Sgs1 and replication dependent mechanism (s) leading to fragile site instability in S. cerevisiae. We are investigating the correlation between slow replicating regions genome and both DNA breaks and chromosomal rearrangements in particular genomic loci. In a related project and in collaboration with the Laboratory of Dr. Angela Taddei at the Curie Institute in Paris we are determining if fragile sites have characteristic dynamics and nuclear position taking a microscopy approach.

S phase checkpoints and Mec1-Ddc2 There are different types of DNA stress during S phase that can elicit activation of Rad53. The replication checkpoint becomes activated when the progression of the replication fork is stalled - as is the case after exposure to hydroxyurea (HU) . This checkpoint is also activated as a consequence of forks colliding with damaged DNA or aberrant DNA structures.The intra-S-phase checkpoint is activated by DSBs generated in genomic loci outside the active replicon region in S phase. The key feature of the DSB-induced intra-S-phase checkpoint that distinguishes it from replication-dependent checkpoints is that it does not require an active replication fork for checkpoint signal initiation. During the checkpoint response Mec1-Ddc2 (human ATR-ATRIP) activates Rad53 and is also recruited to both DSBs and stalled replication forks where it stabilizes components of the replisome. This recruitment is absolutely essential for survival of the cell, and in the case of stalled forks a loss of Mec-Ddc2 results in irreversible fork collapse. In mammals the affinity of ATRIP for RPA suggests a model in which ATR-ATRIP is recruited to DSBs through interaction with RPA-bound single-stranded DNA (ssDNA). In support of this model a mutant allele of RPA, rfa1-t11 was shown to have major defects in the binding of ATP-ATRIP to RPA- ssDNA in vitro and in yeast the same rfa1-t11 mutation showed a significant loss of Mec1-Ddc2 binding at the site of a DSB. Interestingly however this rfa1-t11 mutation has no effect on the recruitment of Ddc2 to HU-stalled forks, underscoring the misconception that Mec1-Ddc2 recruitment to forks and DSB are through identical mechanisms. We are characterizing Mec1-Ddc2 localization to stalled replication forks and the factors involved in its recruitment.

 

Our research is supported by the following granting agencies