Mechanism and regulation of chromatin remodeller, RSC

RSC is a multiprotein complex that remodels the structure of chromatin and in turn regulates processes like transcription. The mechanism by which RSC remodels a nucleosome is still unclear. Although bulk solution experiments have added a growing body of evidence to the mechanism by which RSC remodels a nucleosome, the mechanistic properties of the remodeler is still under debate. In this work, we have constructed a system to study the mechanistic properties of a single molecule of RSC on a naked DNA template in real time and obtain its properties like rate of translocation, processivity, force generated by the translocase and rate of ATP turnover. The SWI/SNF family of chromatin remodeling complexes contains conserved Actin-related proteins (ARPs). Genetic and biochemical evidence suggests that Sth1 might be regulated by two Actin-Related Proteins, ARP7 and ARP9. Genome wide suppressors of ARPs mapped to two domains in Sth1 called the post-HSA (Helicase/ Sant associated) domain and ATPase domain. In this work, dominant negatives of Sth1 were obtained that also localized to the post-HSA (Helicase/ Sant associated) domain. This suggests that the post-HSA domain might be involved in the regulation of Sth1. Sth1 (301-1097) construct containing the suppressor mutations co-purified with ARPs were compared for their ATPase activity and KM with wild type Sth1 (301-1097) Arps complex. The mutants showed higher ATPase activity than wild type, which was not due to their increased affinity for DNA. iv Based on the fact that the loop formed by the action of a remodeler is negatively supercoiled, in this work, we also developed an assay to assess the torsion generated by ATP dependent chromatin remodelers on naked DNA. TetR-Sth1 (301-1146) fusion co-purified with ARPs successfully produced torsion in an ATP dependent manner as assayed by this assay. This assay also depends on the action of by E. coli topoisomerase I, which specifically relaxes negative supercoils and on TetR-TetO interaction which serves to anchor the motor protein and thus constrains the loop formed by it.