GPMLS Lectures - Dr. Sarah Shammas & Dr. Sebastian Deindl

Dr. Sarah Shammas, MRC career development fellow at the Department of Biochemistry at Oxford University and Dr. Sebastian Deindl, Professor of Molecular Biophysics at the Department of Cell and Molecular Biology at Uppsala University, EMBO Young Investigator and Wallenberg Academy Fellow will give lectures in the GPMLS lecture series.

The talk of Dr. Sarah Shammas is titled "Impact of residual structure in DNA binding by disordered DNA binding domains".

Abstract: Transcription factors are heavily enriched in protein disorder, particularly in their activation domains and linker regions. However there are large classes of eukaryotic transcription factors with disordered DNA binding domains, including the basic zippers (bZIPs). bZIPs are disordered in isolation but fold into helices upon DNA binding. One such bZIP is Cyclic AMP Response Element Binder (CREB), which is involved in regulation of multiple cellular processes including proliferation, memory formation, and maintaining the circadian rhythm. We performed kinetic and structural studies of CREB bZIP and a set of designed Ala-Gly mutants to perform the first Phi-value analysis for coupled folding and binding of a protein binding to DNA. We reveal the transition state is disordered and dynamic, but find no evidence of an advantage of protein disorder in binding (fly-casting). We go on to examine the interaction with non-specific DNA; whilst specificity depends on pre-existing residual structure within the DNA binding region, target search efficiency is increased by disorder.

The talk of Professor Sebastian Deindl is titled "Massively parallel analysis of single-molecule dynamics on next generation sequencing chips".

Abstract: Single-molecule techniques are ideally poised to characterize complex dynamics but are typically limited to investigating a small number of different samples. However, a large sequence or chemical space often needs to be explored to derive a comprehensive understanding of complex biological processes. In my talk I will describe our newly developed method, MuSCLe, that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of the dynamics of millions of individual molecules covering thousands of distinct sequences. I will discuss how we comprehensively profiled the sequence dependence of DNA hairpin dynamics and Cas9-induced target DNA unwinding/rewinding dynamics. For Cas9, the ability to explore a large sequence space allowed us to identify a number of target sequences with unexpected behaviors that cannot be explained by canonical base pairing. We envision that MuSCLe will enable the mechanistic exploration of many fundamental biological processes.