Structural dynamics of E. coli single-stranded DNA binding protein reveal DNA wrapping and unwrapping pathways

• Published in: eLife Vol. 4
• Main Authors: Suksombat, Sukrit, Khafizov, Rustem, Kozlov, Alexander G, Lohman, Timothy M, Chemla, Yann R
• Format: Journal Article
• Language: English
• Published: England eLife Sciences Publications, Ltd 25.08.2015; eLife Sciences Publications Ltd
• Subjects: Biophysics and Structural Biology; DNA, Bacterial - chemistry; DNA, Bacterial - metabolism; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; energy landscape; Escherichia coli - chemistry; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Kinetics; Microscopy, Fluorescence; Nucleic Acid Conformation; optical tweezer; Optical Tweezers; Protein Binding; Protein Conformation; protein-nucleic acid interaction; single molecule; single stranded DNA binding protein; energy landscape; protein-nucleic acid interaction; single molecule; single stranded DNA binding protein; optical tweezers; structural biology; biophysics; E. coli
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.08193

Escherichia coli single-stranded (ss)DNA binding (SSB) protein mediates genome maintenance processes by regulating access to ssDNA. This homotetrameric protein wraps ssDNA in multiple distinct binding modes that may be used selectively in different DNA processes, and whose detailed wrapping topologies remain speculative. Here, we used single-molecule force and fluorescence spectroscopy to investigate E. coli SSB binding to ssDNA. Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated. The data allow us to construct an energy landscape for the ssDNA-SSB complex, revealing that unwrapping energy costs increase the more ssDNA is unraveled. Our findings provide insights into the mechanism by which proteins gain access to ssDNA bound by SSB, as demonstrated by experiments in which SSB is displaced by the E. coli recombinase RecA. The DNA double helix consists of two strands coiled around each other. However, there are many instances when DNA must be separated into its individual strands—for example, when the DNA sequence needs to be copied. These single-stranded structures are highly prone to damage. For protection, the single-stranded DNA can wrap around single-stranded DNA binding (SSB) proteins, which also control how other maintenance proteins interact with the DNA. SSB proteins from the bacteria species Escherichia coli wrap single-stranded DNA into a variety of topologies known as binding modes. By using a technique that uses a laser to exert forces on an individual DNA molecule, Suksombat et al. unraveled DNA from a single SSB protein. This revealed that the unraveling occurs in a series of steps that correspond well to the known binding modes. These steps also provide the energies required to unravel the single-stranded DNA. Further experiments showed that SSBs can slide along DNA without having to change their binding mode. The unraveling and sliding mechanisms are likely to be used by other proteins to gain access to DNA coated with SSBs. The next step is to understand how SSBs interact with these other proteins, and how their various wrapping configurations affect this interaction.