DNA replication has an important place in cancer biology: the mutations that cause cancer occur primarily during DNA replication and many of the drugs that are used to treat cancer work by interfering with DNA replication. We are interested in understanding the mechanism of DNA replication, how DNA replication is regulated during the cell cycle and how DNA replication is regulated in response to DNA damage. Much of our progress in 2008 has focussed on DNA damage responses and DNA replication.
DNA damage checkpoints and replication forks
Eukaryotic cells employ specialised surveillance mechanisms called 'checkpoints' to preserve both viability and genome integrity when confronted with endogenous or exogenous agents that interfere with DNA replication. The loss of checkpoint pathways can lead to genomic instability and thereby promote carcinogenesis. We previously found a role for DNA damage checkpoints in maintaining the stability of replication forks that encounter DNA damage. We provided evidence that this role explains why checkpoint mutants are hypersensitive to many DNA damaging agents.
The DNA damage checkpoint involves a protein kinase cascade and, in Saccharomyces cerevisiae, the central checkpoint protein kinases are the ATR homolog Mec1 and its downstream effectors, Chk1 and the Chk2 homolog Rad53. In response to S-phase perturbations, Mec1 is recruited to stalled DNA replication forks where it is required to phosphorylate and activate Rad53. How these kinases then act to stabilise DNA replication forks has been somewhat mysterious, but is likely to have important implications for understanding how genomic instability is generated during oncogenesis and how chemotherapies that interfere with DNA replication might be improved.
During this past year we showed that the sensitivity of rad53 mutants to a variety of DNA damaging agents can be almost completely suppressed by deletion of the EXO1 gene. Deletion of EXO1 also suppressed DNA replication fork instability in rad53 mutants. Deletion of EXO1, however, was completely ineffective in suppressing both the sensitivity and replication fork breakdown in mec1 mutants indicating that Mec1 has a genetically separable role in replication fork stabilisation from Rad53. This analysis also showed that the second downstream effector kinase, Chk1, which had not previously been implicated in budding yeast S phase responses to DNA damage, can stabilise replication forks in the absence of Rad53 (Figure 1). Our results reveal previously unappreciated complexity in the downstream targets of the checkpoint kinases and provide a framework for elucidating the mechanisms of DNA replication fork stabilisation by these kinases. Down regulation of DNA damage checkpoints is believed to occur in many cancers and may help to explain why many cancers are responsive to chemotherapies that act by damaging DNA. Our results suggest that the loss of function of nucleases like Exo1 may represent a novel mechanism by which cancer cells become resistant to these drugs.
Factors influencing DNA double strand break response
Double strand breaks (DSBs) in DNA are amongst the most dangerous of chromosomal lesions, and can lead to cell death and genomic rearrangements. Two major pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR), compete for the repair of DSBs. During the first step of HR, breaks undergo nucleolytic degradation of their 5'-ending strands, a process known as resection. This generates 3'-ended single-stranded tails, which are required for the downstream events in HR. Resection also generates DNA end structures that are not used efficiently in NHEJ thus contributing to the switch between repair pathways.
The choice between NHEJ and HR is also regulated by cyclin-dependent kinases (CDKs), and is therefore influenced by cell cycle stage. Cells are proficient for NHEJ in G1 when CDK activity is low, but not in G2/M, when CDK activity is high and HR is predominant. The molecular mechanism underlying these CDK-dependent effects is important but still obscure. In this past year, we described a novel quantitative assay to analyse DSB processing in the budding yeast, Saccharomyces cerevisiae. Consistent with previous work, we found evidence for extensive resection (>10 kb) from a DSB induced by the site specific HO endonuclease. However, our quantitative assay showed that only a small fraction of breaks are resected to this extent. This, together with previous genetic analysis suggests that resection may not be the only pathway for generating the ssDNA required for HR. We suggest that unwinding by an unidentified DNA helicase may be important for this. Our analysis also provided the first evidence for significant instability of the 3' ssDNA tails. Thus, resection is not limited to the 5' strand: the 3' strand is also degraded, although this resection lags behind the 5' strand resection. Resection of the 3' strand might aid recombination or single strand annealing by ensuring there is a 3' end close to the region of invasion/annealing. We also showed that both DSB resection and checkpoint activation are dose-dependent, especially during the G1 phase of the cell cycle. During G1, processing near the break is inhibited by competition with NHEJ but extensive resection is regulated by an NHEJ-independent mechanism. DSB processing and checkpoint activation are more efficient in G2/M than in G1 phase; however, we found that both processes are by far most efficient during S phase. We showed that this enhanced response requires that DNA replication forks encounter the break.
Our work in 2008 has helped identify important connections between DNA replication and DNA damage responses. This work was done primarily in budding yeast, and it will be important to examine these links in human cells to determine whether they have roles in cancer biology.
Figure 1. Model describing the role of DNA damage checkpoint kinases in regulating DNA replication fork stability after encountering a lesion (red X) in budding yeast. See text for details.
For a list of refereed research papers, see Publications (in navigation on left).
