Objective 1: Deciphering the role of WH domain in determining substrate selectivity of RECQ1
RecQ helicases are a highly conserved family of genome maintenance proteins having diverse roles in nucleic acid metabolic pathways. The prototypical member of this family is the Escherichia coli RecQ helicase (EcRecQ). Five members of the RecQ family have been found in human: BLM, RECQ1, RECQ4, RECQ5 and WRN. The catalytic core of RecQ includes the helicase domain and the RQC domain, the latter consisting of Zn binding (Zn) domain and Winged helix (WH) domain. There are several reports of differences in substrate specificity between RecQ family members. The substrate specificity of human RECQ1 is distinct from that of EcRecQ helicase. A comparison of the crystal structures of EcRecQ and RECQ1 reveals that the WH domain is positioned very differently in the two helicases. This difference in domain orientation may reflect alternative conformations available to each protein. The WH domain of the E. coli protein is positioned perpendicular to the D1–D2 domains, whereas in the human RECQ1 the WH domain lies directly beneath the helical hairpin portion of the Zn domain, creating a more elongated linear molecule (Pike et al., 2009). Although human RECQ1 has maximum sequence similarity with EcRecQ, and both of them share similar size and domain organization, their distinct substrate specificities may arise from their differences in the relative orientation of the WH domain. It would be tempting to hypothesize that the structure and conformation of the WH domain would be the determinant for selecting and binding to the substrates.
Objective 2: Studying the dynamics of replication fork progression in presence of human RECQ1 and Replication Protein A
RecQ helicases interact with a number of protein partners in the cells in various DNA metabolic pathways. A key cellular partner of RecQ helicases is Replication Protein A (RPA), a single-stranded DNA binding protein involved in DNA replication, repair, recombination, telomere maintenance and DNA damage response pathways. RPA is a heterotrimeric protein complex comprising of 3 subunits – RPA70, RPA32 and RPA14. RECQ1 restores replication forks reversed by Topoisomerase I inhibition via its ATPase and branch migration activities. Stabilization and restoration of stalled forks is significant for cell survival. Hence cancerous cells bearing BRCA1/2 mutations targeted with PARP inhibitors showed strong synthetic lethality as the combined loss of PARP and BRCA1/2 caused severe defects in fork stability and consequent DSBs in the DNA (Bryant et al., 2005). However, resistance to PARP inhibitors has been shown to develop due to stabilization of regressed forks in BRCA deficient cancer cells. Moreover, DNA damages induced by chemotherapeutic agents like cisplatin and topotecan are efficiently repaired by fork restoration, thereby reducing the efficacy of the drugs (Lord and Ashworth, 2013). In this regard, RECQ1 could be a promising target in combination with Topoisomerase I posisons to synergistically induce fork reversal and inhibit fork restoration leading to synthetic lethality (Berti et al., 2013). Hence it is essential to understand the molecular mechanism of replication fork restoration by RECQ1 in order to formulate more efficient chemotherapeutic strategies.
