'Polymerase Switching during Translesion DNA Synthesis and Quantitative Protein Labeling for Single-molecule Imaging"

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Date/Time:Thursday, 31 Jan 2013 from 3:10 pm to 4:00 pm
Location:Physics 0005
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Xinghua Shi (University of Illinois, Urbana-Champaign)

DNA in a living cell is often under the threat of various damaging agents such as UV irradiation. Since the discovery that organisms in all three domains of life possess specialized DNA polymerases known as translesion polymerases, these enzymes and the process of DNA replication across certain persisting lesions have attracted widespread interest. These proteins, although robust in damage tolerance, are error-prone in general. Therefore, their access to DNA needs to be well regulated to ensure genome integrity and stability. Essential to DNA replication and translesion synthesis is a ring-shaped protein called sliding clamp. Based on the clamp’s oligomeric structure and multivalent potential, it was proposed in the “tool belt†model that more than one polymerase can bind to the same clamp molecule simultaneously during lesion bypass. Here, I will present our findings on the transient interactions and switching between a replicative polymerase and a translesion polymerase on the clamp from an archaeal species, and examine several relevant models including the “tool beltâ€. We demonstrate that the replicative polymerase can not only co-localize transiently with the translesion polymerase, but get removed rapidly from the clamp on a damaged DNA by excess translesion polymerase, using the latter’s polymerase domain and clamp-interacting motif through a synergistic mechanism. Direct visualization of these transient protein-protein interactions was made possible for the first time by single-molecule imaging techniques, including fluorescence resonance energy transfer (FRET), and a new protein labeling method we developed recently.
A major hurdle for molecular mechanistic studies of many proteins was the lack of a general method for fluorescence labeling with high efficiency, specificity and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieve fast, site-specific labeling of a protein with ~100% efficiency while maintaining the biological function. An aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in single-molecule pull-down analysis. The unique power of this method is shown in the single-molecule study above on the interactions and switching between DNA polymerases on the sliding clamp.

Dr. Xinghua Shi is a postdoctoral fellow from Taekjip Ha's lab at the University of Illinois, Urbana-Champaign and Howard Hughes Medical Institute. His work in Illinois has been focused on 1) the mechanistic understanding of polymerase switching during translesion DNA synthesis, and 2) the development of quantitative protein labeling through an aldehyde tag for single-molecule imaging. Prior to Illinois, Dr. Shi obtained his Ph.D. in Physical Chemistry from Stanford University in 2007, where he in Steve Boxer's lab on ultrafast fluorescence spectroscopy and excited-state dynamics in green fluorescent proteins. Xinghua was originally from China and received his B.S. in Chemical Physics from the University of Science and Technology of China. He enjoys spending his leisure time with his family and watching college football.