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Title:Binding configurations of single-stranded DNA binding protein and their influence on DNA recombinase
Author(s):Suksombat, Sukrit
Director of Research:Chemla, Yann R.
Doctoral Committee Chair(s):Ha, Taekjip
Doctoral Committee Member(s):Aksimentiev, Aleksei; Myong, Su-A
Department / Program:Physics
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Optical Tweezers
Single-stranded DNA Binding Protein
Single-stranded DNA binding (SSB)
Deoxyribonucleic Acid (DNA)
Optical Traps
Abstract:DNA inside a cell is continuously damaged through multiple mechanisms including environmental exposure to radiation, chemical agents, or UV light. Certain products of the cell's own metabolism, such as reactive oxygen species, can also damage the DNA. In the worst-case scenario, this damage results in double-stranded DNA (dsDNA) breaks. Double-stranded DNA breaks are lethal, and difficult to repair, with potential complications from genome rearrangement. To prevent this genetic instability, a cell can utilize a homologous chromosome as a template to accurately repair DSBs. This process is called homologous recombination. Homologous recombination begins when an enzyme complex binds to a blunt end of a dsDNA break. The complex unzips the dsDNA through its helicase activity, and simultaneously cleaves the newly-generated 5' end of the ssDNA. This process leaves the remaining ssDNA strand exposed to the surrounding environment and prone to nucleolytic and chemical attacks. Cells have evolved single-stranded DNA binding (SSB) proteins to wrap and protect this ssDNA. In E. coli, SSB is known to wrap ssDNA in a variety of binding configurations, or modes. Three different binding modes, (SSB)65, (SSB)56, and (SSB)35, which wraps 65, 56, and 35 nucleotides (nt) respectively, have been observed in vitro Previous studies have suggested that SSB binding in different modes may exhibit different levels of binding cooperativity. SSBs in the (SSB)65 binding mode form isolated clusters (limited cooperativity), while SSBs in the (SSB)35 binding mode form long filaments (unlimited cooperativity). These different levels of binding cooperativity have been proposed to be used selectively in different DNA metabolic processes, including DNA replication, recombination, and repair. In homologous recombination, recombinase RecA must bind and form nucleoprotein filaments on the ssDNA, in direct competition with SSB. Prior studies have shown that RecA is capable of forming filaments on ssDNA wrapped by SSBs in the (SSB)65 binding mode, but filament formation on ssDNA wrapped by SSBs in the (SSB)35 binding mode is inhibited. Recent single-molecule studies have been conducted to investigate this competitive process, but the detailed mechanisms remain unclear. Here, we use high-resolution optical tweezers with simultaneous fluorescence microscopy to observe directly the activity of ssDNA-SSB, ssDNA-RecA, and ssDNA-SSB-RecA complexes under tension, and characterize their mechanical properties. The instrument allows us to simultaneously probe and visualize the interactions of RecA and SSB with ssDNA in real time and with nanometer resolution. We confirm that individuals SSBs bind and compact ssDNA in discrete modes. Under low tension (1-3 pN), a single SSB wraps ssDNA in the (SSB)65 or (SSB)56 binding mode. At higher tension (4-8 pN), SSB exhibits transient wrapping-unwrapping, switching between the (SSB)56, (SSB)35, and (SSB)17 wrapping modes. When multiple SSBs are present on the ssDNA, the SSBs form isolated clusters in those solution conditions that favor the (SSB)65 binding mode. The configuration of the SSBs changes to a long and stable filament when solution conditions that favor the (SSB)35 binding mode are used. In the absence of SSB, RecAs nucleate filament rapidly on ssDNA. The nucleation rate of RecA is slowed down by several times when RecA is added to ssDNA coated with isolated clusters of SSBs in the (SSB)56 mode. The nucleation rate of RecA decreases further when long and stable filaments of SSBs in the (SSB)35 binding mode are present on the ssDNA. The same experiments also demonstrate that RecA is capable of removing these SSBs from the ssDNA in a step-wise manner. Our results reveal the importance of SSB binding modes and their oligomerization to DNA recombination, and further confirm that (SSB)65/(SSB)56 binding modes are more likely to facilitate the activity of recombinase RecA during the DNA repair. The (SSB)35 binding mode, on the contrary, inhibits RecA filament formation, and is believed to not play an important role in this recombination process.
Issue Date:2015-04-22
Rights Information:Copyright 2015 Sukrit Suksombat
Date Available in IDEALS:2015-07-22
Date Deposited:May 2015

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