Research Overview

Summary

In response to DNA damage, conserved checkpoint surveillance mechanisms trigger a cascade of events that coordinate cell cycle arrest with DNA repair; the cell cycle only reinitiates once the offending lesion has been removed and DNA integrity restored. A failure to invoke the DNA damage response (DDR), whether at the level of checkpoint signaling or DNA repair, results in persistent DNA damage and has catastrophic consequences for genome stability and cancer susceptibility. Over the past decade it has become increasingly apparent that many inherited cancer predisposition syndromes occur as a consequence of mutation in critical DDR genes. A comprehensive understanding of the DDR should therefore provide important insights into tumorigenesis and may present opportunities for therapeutic intervention.

The major threat to genome stability in normal cells is the failure to correctly respond to DNA damage during S-phase. Of particular interest to the lab has been the discovery that the BRCA and Fanconi anemia (FA) pathways facilitate DNA repair in S-phase. While FA is a multi-genic cancer predisposition disorder, and little is known about how it functions in DNA repair, the BRCA pathway is better understood. BRCA1 and BRCA2 are two tumor suppressor genes in this pathway, implicated in breast and ovarian cancer. Both genes mediate by homologous recombination (HR). Interestingly, the BRCA and FA pathways seem to be linked: the BRCA1 associated helicase BRIP1 is mutated in FA-J patients and BRCA2 is mutated in the FA-D1 subtype of the disease.

Over the past few years the lab has established that BRCA and FA pathways are functionally conserved in the nematode C. elegans. To examine the contribution of the BRCA and FA pathways in repair of DNA damage in S-phase we have developed C. elegans as a model system for studying the repair of lesions that block DNA replication, including DNA interstrand cross-links (ICLs). Our studies of ATL-1 (C. elegans ATR) and the single Rad51 paralog in C. elegans have provided unexpected insights into the sensing and repair of replication blocking lesions and DSBs. By dissecting the BRCA and FA pathway in C. elegans we have discovered new connections between the S-phase checkpoint and FA and HR-mediated repair; these include new factors that impact on BRCA and FA pathways in both C. elegans and mammalian cells, a novel S-phase checkpoint gene and a novel helicase that acts as an antagonist of homologous recombination. The aim of our current and future work is to define further how DNA damage is sensed and repaired during S-phase. Considerable effort is now being made to extend our findings in C. elegans to mouse models and human cell culture, which we hope will provide further insight into the fundamental roles performed by DDR pathways in human disease.

THE DNA DAMAGE RESPONSE

The DDR requires the orchestration of highly specialised cell cycle checkpoints, each of which must be rapidly activated following the detection of damaged DNA. Such checkpoints operate at the G1/S, intra-S and G2/M boundaries of the cell cycle and are controlled by the ATM/Chk2 and ATR/Chk1 pathways. The intra-S-phase checkpoint, which is under the control of the ATR/Chk1 pathway, prevents the collapse of stalled replication forks and thus plays an essential role in maintaining genome integrity during S-phase. Upon detection of DNA damage the intra-S-phase checkpoint also co-ordinates cell cycle arrest with DNA repair. The functional importance of checkpoints and repair pathways in maintaining genome stability is highlighted by their conservation throughout eukaryotes and by the many human disease syndromes that result from defects in DDR factors. It is therefore important to understand these complex pathways at the molecular level to further our knowledge of cancer progression and its treatment.

DSB REPAIR

DNA double strand breaks (DSBs) represent one of the major threats to genome integrity. Eukaryotic cells possess at least three pathways for DSB repair: non-homologous end joining (NHEJ), single-strand annealing (SSA) and homologous recombination (HR). In contrast to NHEJ and SSA, which are intrinsically error-prone in nature, HR is the predominant mechanism employed by cells to accurately repair DSBs in S and G2 phases of the cell cycle. HR is also fundamentally important for a variety of DSB-initiated DNA transactions such as those occurring during meiotic recombination, V(D)J recombination and mating type switching.

During evolution HR has also taken on an increasingly important role in DNA replication. While HR is dispensable for DNA replication in yeast, it is essential for completion of S-phase in complex eukaryotes, where it is presumed to be important for re-generation of stalled or collapsed replication forks. At the mechanistic level, HR is initiated by nucleolytic processing of the DSB, which generates recombination-proficient 3' single stranded DNA (ssDNA) overhangs that are rapidly bound by replication protein A (RPA). Rad51, a key recombinase enzyme and the eukaryotic counterpart of RecA, displaces RPA-ssDNA complexes to form a helical nucleoprotein filament. It is within the context of the nucleoprotein filament that Rad51 is able to search for an intact homologous template and then catalyzes invasion of the ssDNA into an intact donor sister chromatid or homologous chromosome to form a joint molecule. The resulting joint molecule acts as a primer for DNA synthesis to extend the heteroduplex DNA that, following further processing and resolution of the joint DNA molecules, leads to repair of the DSB and restoration of DNA integrity.

BRCA GENES

Of particular interest has been the realization that the hereditary breast and ovarian cancer tumour suppressor genes BRCA1 and BRCA2 both function in HR-mediated DSB repair. Although BRCA1 and BRCA2 both co localize with Rad51 at repair foci and are both required for recruitment of Rad51 to DSBs, their respective roles in promoting HR are very different. BRCA1 exists as a heterodimer with the structurally related protein BARD1 and together they facilitate S-phase and G2/M checkpoint responses to DNA damage. In this regard, BRCA1/BARD1 appear to function as a checkpoint mediator, similar to Mdc1, 53BP1 and Claspin, which collectively stimulate interactions between ATM/ATR and their substrates by mediating the assembly of multi-protein complexes at and around repair sites. Indeed, activation of the Chk1 kinase by ATR-dependent phosphorylation is compromised in the absence of BRCA1/BARD1. In turn, Chk1 not only signals to the cell cycle but also promotes HR-mediated DSB repair through direct phosphorylation of Rad51. Therefore it appears that BRCA1/BARD1 deficiency compromises checkpoint signalling and HR-mediated DSB repair due to a reduced capacity to activate Chk1.

What remains unclear is how BRCA1/BARD1 performs this function. An emerging theory is that the E3 ubiquitin (Ub) ligase activity of the RING domains within the BRCA1/BARD1 heterodimer might be involved. This activity is clearly important physiologically as ubiquitylation is abolished by a Tumor-derived mutation (C61G) in one of the conserved cystine residues within the BRCA1 RING. In the last few years my lab and others have demonstrated that the E3-Ub ligase activity of the BRCA1/BARD1 heterodimer is activated by the checkpoint and is responsible for ubiquitylation of factors at sites of DNA damage. The identity of these factors has remained elusive until very recently and the consequence of their ubiquitylation is still unknown.

In contrast to the signalling role performed by BRCA1, BRCA2 directly binds to and regulates Rad51 in HR-mediated DSB repair. Human BRCA2 binds to Rad51 through BRC repeat domains in the central portion of the protein and a second distinct site in the C-terminus that is regulated by Cdk1 phosphorylation. In addition, structural analysis of the C-terminal region of BRCA2 revealed that BRCA2 binds to ssDNA via three oligonucleotide-oligosaccharide-binding folds. The presence of Rad51 binding and ssDNA binding activities in BRCA2 has led to the simple model that BRCA2 promotes HR-mediated DSB repair by binding to Rad51 and targeting it to 3' ssDNA overhangs at resected DSBs. In recent years, BRCA2 homologs have been identified in most eukaryotes, with the exception of budding and fission yeasts. While conservation between species is weak, BRCA2 homologs possess at least one BRC motif and one ssDNA-binding domain. Recent biochemical studies of BRCA2 homologs also support a role for BRCA2 in stimulating Rad51 activities.

FANCONI ANEMIA

Further links between HR and human disease have been revealed by the findings that BRCA2 and its associated partner protein PALB2 are mutated in the D1 and N complementation groups of Fanconi anaemia. So far, 13 genetic complementation groups of FA (A, B, C, D1, D2, E, F, G, I, J, L, M and N) have been defined, mostly by somatic cell fusion studies and sensitivity to MMC. Eight of the cloned FA proteins (A, B, C, E, F, G, L and M) are believed to constitute a multi-subunit nuclear complex termed the FA core complex, whereas FANCD1/BRCA2, FANCD2, FANCJ/BRIP and FANCN/PALB2 are thought to function downstream. A major function of the FA core complex is to mono-ubiquitylate FANCD2 and FANCI at specific lysine residues during S-phase or in response to various DNA damaging agents. The ubiquitylation activity of the FA core complex is provided by the FANCL subunit, which encodes a PHD/RING containing Ub-ligase, whereas de-ubiquitylation of FANCD2 following ICL repair is mediated by USP-1. At the cellular level, FANCD2 and FANCI mono-ubiquitylation is important for chromatin association and recruitment to DNA damage sites where it forms nuclear foci that co-localize with several DNA repair proteins, including BRCA1 and BRCA2/FANCD1. Current evidence supports a role for FANCD2 and the FA pathway in orchestrating lesion repair via homologous recombination (HR) and/or translesion bypass pathways. However, the precise mechanism through which the FA pathway and mono-ubiquitylation of FANCD2/FANCI promotes DNA repair remains unclear.

INSIGHTS FROM C. ELEGANS

Since the BRCA and FA pathways are not conserved in yeast, elucidation of their respective functions and their affect on HR and other DNA repair pathways would be greatly enhanced by the identification of conserved BRCA and FA pathways in a simple model system. My postdoctoral work with Dr. Marc Vidal established C. elegans as a model system for the analysis of DDR pathways [31]. Over the past five years, my lab has identified functional homologs of BRCA1, BARD1, BRCA2/FANCD1, FANCD2 and FANCJ in C. elegans. Through a combination of biochemistry and genetics, we have established C. elegans as the simplest genetically tractable organism that can be used for the study of BRCA and FA pathways. Although studies of the DDR remain largely unexplored in this organism, C. elegans presents some unique advantages that we can exploit to gain new insights into the function of the BRCA and FA pathways. These include forward and reverse genetic approaches and the spatial organization of mitotic and meiotic prophase cells within the germline (Fig. 1). The distal end of the germline comprises a compartment of mitotically proliferating nuclei that are followed, more proximally, by cells in progressive stages of meiosis I. This spatial restriction of SPO-11 induced meiotic DSBs allows separation of factors required for repair of meiotic DSBs from factors required for repair of replication-induced DSBs in mitotic cells. For this reason, C. elegans is advantageous as a model to study repair at both conventional DSBs and replication fork barriers (RFBs), such as poly G/C tracts and DNA interstrand cross-links (ICLs).

To complement the power of forward and reverse genetic approaches in C. elegans with biochemical studies, my lab developed tandem immunoaffinity purification methods for purifying protein complexes from worms [34, 35]. The major advantages of our system are that: complexes can be purified from whole animals rather than a transformed cell line; complexes can be purified under native conditions that allow subsequent biochemical analysis; and complexes can be purified from multiple cell types and different developmental stages. We have established that identification of protein complexes using this system, coupled with validation using reverse genetic approaches, provides a powerful combination for understanding gene function. This approach has been successfully employed to purify factors associated with C. elegans BRCA1/BARD1 and FANCD2.

Our published work has demonstrated that the BRCA and FA pathways in C. elegans are simplified compared with their vertebrate counterparts, yet many of the fundamental properties of these pathways are conserved in C. elegans. By exploiting the genetics and biochemistry of the C. elegans system, we have provided novel insights into the function and regulation of these pathways in both C. elegans and human cells, and have begun to develop C. elegans as a model system for studying repair of DNA lesions in S-phase. Importantly, our approach in C. elegans has led to the identification of new components that impact on BRCA and FA pathways in human cells. A major effort of the lab is to extending our findings in C. elegans to mouse models and human cell culture, which we hope will provide further insight into the fundamental roles performed by DDR pathways in human disease.