RAD51 paralogs in Fanconi anemia and breast cancer

Fanconi anemia (FA) is a rare autosomal and X-linked recessive chromosome instability syndrome characterized by congenital abnormalities, progressive bone marrow failure, predisposition to acute myelogenous leukemia and malignancies. FA proteins are involved in the repair of inter strand crosslinks (ICLs) and so far about 19 genes have been identified to play a role in FA pathway of ICL repair. RAD51 and RAD51 paralogs are important for HR and in the maintenance of genome stability. Despite the identification of five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) over a decade ago, the molecular mechanism(s) by which RAD51 paralogs regulate HR and genome maintenance remains obscure. In a striking new finding, germline mutations in RAD51C have been identified to cause FA-like disorder and breast and ovarian cancers. Our work showed that RAD51C indeed participate in the FA-pathway of ICL repair. RAD51C plays at the HR step of ICL repair. For the first time we showed that RAD51C controls intra-S-phase checkpoint via CHK2 activation. Using pathological mutants we demonstrated that RAD51C distinctly regulate DNA damage signaling and repair. We also showed that hypomorphic RAD51C pathological mutants can be specifically targeted in a synergistic approach by using low dose of PARP inhibitor and enhancing NHEJ with low dose of IR.

RAD51 paralogs in genome maintenance

The phenomenon of chromosomal instability (CIN) is a hallmark of nearly all cancer types. CIN develops at early stages of cancer, and replication stress in the form of fork stalling is proposed to be the prominent driving force for this instability. The link between replication stalling to tumor development is more appreciated after the observation that oncogene activation induces replication stress. The BRCA1, BRCA2, PALB2 and FA pathway proteins such as FANCD2 are known to function in DSB repair by HR. Notably, in addition to their recombinational repair role, these proteins have been recently shown to participate in replication fork protection. Mammalian RAD51 paralogs are implicated in the repair of collapsed replication forks by homologous recombination (HR). However, their physiological roles in replication fork maintenance prior to fork collapse remain obscure. We have recently showed that RAD51 paralogs particpate in short-term replicative stress devoid of DSBs. We show that RAD51 paralogs localize to nascent DNA and common fragile sites upon replication fork stalling. Strikingly, RAD51 paralogs deficient cells exhibit elevated levels of 53BP1 nuclear bodies and increased DSB formation, the latter being attributed to extensive degradation of nascent DNA at stalled forks. RAD51C and XRCC3 promote the restart of stalled replication in an ATP hydrolysis dependent manner by disengaging RAD51 and other RAD51 paralogs from the halted forks. Notably, we find that Fanconi anemia (FA)-like disorder and breast and ovarian cancer patient derived mutations of RAD51C fails to protect replication fork, exhibit under-replicated genomic regions and elevated micro-nucleation. Collectively, RAD51 paralogs prevent degradation of stalled forks and promote the restart of halted replication to avoid replication fork collapse, thereby maintaining genomic integrity and suppressing tumorigenesis. Further studies are in progress to understand the molecular mechanism(s) of RAD51 paralogs mediated genome maintenance functions.

RAD51 paralogs in DNA damage responses

Mutations in any of the RAD51 paralog leads to impaired DSB repair by HR, chromosomal aberrations and embryonic lethality. The mechanism(s) by which RAD51 paralogs maintain genome integrity and suppress tumorigenesis is largely unclear. In response to DNA damage, activated ATM/ATR kinase phosphorylates various substrates preferentially at SQ or TQ motifs and induces DNA damage response. This response controls cell cycle checkpoint, DNA repair, apoptosis or senescence. Our work demonstrated that XRCC3 S225 is a phosphorylation target of ATM/ATR kinase and this phosphorylation is essential for HR mediated DSB repair and for the execution of intra-S-phase checkpoint. We are interested in understanding whether other RAD51 paralogs are phosphorylation targets of ATM/ATR kinases and their role in DNA damage responses and genome maintenance.

Fanconi anemia proteins in sister chromatid recombination

FA is a genetically heterogeneous disorder and 19 genes (A, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, Q, R, S and T) have been identified to play a role in the FA pathway of ICL repair. Recent studies indicate that the primary function of FA pathway is to activate DNA damage response and the repair of damaged DNA in the S-phase of the cell cycle. In response to DNA damage, activation of FA pathway leads to monoubiquitination of FANCD2 and FANCI in S-phase. The monoubiquitinated FANCD2 and FANCI specifically localizes with replication and repair foci containing BRCA1, RAD51 and PCNA proteins. These proteins are required for the error free replication of the DNA and repair of damaged DNA during replication by HR. However, the molecular mechanism by which FA protein regulate HR remains obscure. Sister chromatid serves as an ideal template for the repair of DSBs and DSGs that arise during replication. The copied information during sister chromatid recombination (SCR) is accurate. Thus, SCR is potentially an error free pathway for maintaining genome integrity and prevention of cancer. Our lab is interested in understanding the role of FA genes in the SCR pathway of DSB repair.

ATM and ATR kinase in DNA damage response and HR

In response to DNA damage, mammalian ATM and ATR signaling kinases activate a complex network of DNA damage response (DDR) pathway, which coordinates cell cycle checkpoint and DNA repair function. ATM and ATR mutation causes DDR deficiencies, resulting in disorders, such as ataxia-telangiectasia and ATR Seckel syndrome, respectively, which are characterized by developmental defects, DNA damage sensitivity and cancer predisposition. The molecular mechanism by which ATM and ATR kinases sense DNA damage and regulate the downstream signaling cascade, checkpoint activation and DNA repair events are not well understood. The DNA double-strand breaks (DSBs) are the most serious form of DNA damage and unrepaired or misrepaired DSBs leads to genome instability and cancer. The DSBs that are generated in S and G2 phase are preferentially repaired by SCR, an HR pathway that uses neighboring sister chromatid as a template for the repair of DSB. Since the copied information is accurate, SCR is potentially error free. Many genes including ATM and ATR have been implicated in SCR regulation. However, the molecular mechanism by which these kinases regulate SCR is obscure. We are interested in studying the role of ATM and ATR kinases in SCR regulation and SCR mediated gene conversion tracts.

Mycobacterium tuberculosis and DNA repair

Tuberculosis (TB) and AIDS are the major global health crisis, especially in developing countries. Infection with TB is a significant cause of AIDS associated mortality in developing countries. Mycobacterium tuberculosis, the causative agent of TB, can persist for decades in infected individuals in the latent state as an asymptomatic disease and can emerge to cause active disease at a later stage. There is evidence that M. tuberculosis cells are exposed to DNA damaging agents such as reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) generated by host macrophages. Thus, DNA repair pathways and the mechanisms that are involved in the maintenance of genome integrity appear to be an important factor for M. tuberculosis pathogenesis and persistence in the host. Helicases are known to play an important role in DNA replication, repair and recombination. Thus, helicases could be a potential drug target against M. tuberculosis. We are interested to study various helicases and other DNA repair/recombination proteins from M. tuberculosis through genetic, biochemical and biophysical approach. Understanding the role of these proteins could provide new insights into pathogenesis of M. tuberculosis in humans.