Structural biology; nucleic acid regulation; RNAi; molecular recognition; X-ray crystallography
About the Lab
We study the molecular basis of nucleic acid regulatory processes, RNAi and DNA replication in particular. We use the tools of structural biology, biochemistry and biophysics to study proteins and protein complexes associated with these processes to elucidate how they work. X-ray crystallography, EM, and other structural techniques enables us to obtain the three-dimensional structures of these molecular machines. Biochemistry, biophysics and molecular biology allow us to study properties that can be correlated to their function and biology.
The introduction of exogenous double-stranded RNA (dsRNA) into a cell can trigger the gene silencing process called RNA interference or RNAi. Although there has been remarkable progress in unraveling the components of the RNAi machinery, in order to get a complete understanding of these mechanisms we must determine how they work at the molecular level. Therefore, we embarked on structural and biochemical studies of these proteins. By solving the structure of a full-length Argonaute protein, the key component in the RNAi machinery, we identified Argonaute as “Slicer”, the effector enzyme that harbors the small RNAs, e.g. miRNAs and siRNAs, and cleaves the mRNA as directed by the siRNA. These studies enhance not only our understanding of this important pathway, but should also improve the practical use of the RNAi technology as an experimental tool for gene knockdown technology.
Another area of research in the lab is DNA replication. The goal is to understand the molecular motors involved in replication initiation. We have been studying the viral replicative helicase E1 from papillomavirus as a model system to study helicase function and assembly as well as DNA melting and unwinding. We have also been examining the eukaryotic machinery with the Origin Recognition Complex (ORC) as the centerpiece of these studies.
Tocilj, K. F. On, Z. Yuan, J. Sun, E. Elkayam, H. Li, B. Stillman* and L. Joshua-Tor*, Structure of the active form of human Origin Recognition Complex and its ATPase motor module, eLife (2017);6:e20818. doi: 10.7554/eLife.20818.
J. J. Ipsaro, C. Shen C, E. Arai, Y. Xu, J. B. Kinney, L. Joshua-Tor, C. R. Vakoc and J. Shi, Rapid generation of drug resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis, PLoS ONE 12(2) (2017):e0172177. doi:10.1371/journal.pone.0172177
E. Elkayam, R. Parmar, C. R. Brown, J. L. Willoughby, C. S. Theile, M. Manoharan*, L. Joshua-Tor*, siRNA carrying an (E)-vinylphosphonate moiety at the 5’ end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2, Nucl. Acids Res. (2016). doi: 10.1093/nar/gkw1171.