Single Stranded Binding (SSB) proteins are essential in all organisms due to their interactions with ssDNA formed transiently during DNA replication, recombination and repair. Our focus is on E. coli SSB which is the prototypical homotetrameric bacterial SSB. Over the past several years it has become evident that E. coli SSB protein plays a central role in genome maintenance by directly interacting, primarily via its 4 unstructured C-terminal tails, with at least 14 other proteins to bring them to their sites of action on DNA. Yet, how SSB selects among these many proteins (specificity) and what mechanisms are used by these proteins to gain access to SSB-coated ssDNA are not understood. The extremely high affinity of E. coli SSB for ssDNA and its slow dissociation rate from ssDNA would appear to make it difficult for other proteins to gain access to ssDNA. E. coli SSB protein possesses four DNA binding domains and can bind ssDNA in multiple binding modes that differ starkly in their properties, in particular, the number of subunits interacting with ssDNA, which affects ssDNA wrapping around the tetramer, and its inter-tetramer ssDNA binding cooperativity. Our hypothesis is that the different equilibrium and dynamic properties of the multiple SSB-ssDNA binding modes, along with direct SSB-protein interactions, can regulate access of other proteins to the ssDNA. Ensemble thermodynamic approaches will be used to investigate the energetics and specificity of the interactions of SSB with its many SSB interacting proteins (SIPs). We will also examine the kinetics and mechanism by which SIPs bind to the unstructured C-termini of SSB and if SIPs can then be transferred directly to ssDNA. The effects of the different SSB-ssDNA modes and cooperativities on the specificity of EcoSSB binding to these SIPs will be examined. We will use optical and magnetic tweezer approaches to apply force to the ssDNA to selectively populate the different SSB-ssDNA binding modes, which is difficult to accomplish in ensemble studies without changing solution conditions. In this manner we can examine the effects of the different SSB binding modes on the ability of RecA protein and SIPs to gain access to SSB-coated ssDNA under identical solution conditions and thus isolate the effects of the different binding modes. Furthermore, we have recently shown, using single molecule fluorescence approaches that an SSB tetramer is able to diffuse along ssDNA while retaining its high affinity. We will investigate the mechanism of this diffusion and its role in allowing access of other proteins to SSB-coated ssDNA. E. coli SSB is an essential bacterial protein and is involved in all DNA metabolic processes through its protein-protein and protein-DNA interactions, thus our basic studies of EcoSSB can potentially lead to a new target for the development of new antibiotics.
Single Stranded Binding (SSB) proteins are essential in all organisms due to their interactions with single stranded DNA formed transiently during DNA replication, recombination and repair. Our focus is on E. coli SSB which also plays a central role in genome maintenance of this bacterium by interacting with and regulating a myriad of other proteins, yet how SSB accomplishes this is not known. Our basic studies of EcoSSB and its interacting proteins can potentially lead to a new target for the development of new antibiotics.
|Kozlov, Alexander G; Shinn, Min Kyung; Weiland, Elizabeth A et al. (2017) Glutamate promotes SSB protein-protein Interactions via intrinsically disordered regions. J Mol Biol 429:2790-2801|
|Sokoloski, Joshua E; Kozlov, Alexander G; Galletto, Roberto et al. (2016) Chemo-mechanical pushing of proteins along single-stranded DNA. Proc Natl Acad Sci U S A 113:6194-9|
|Waldman, Vincent M; Weiland, Elizabeth; Kozlov, Alexander G et al. (2016) Is a fully wrapped SSB-DNA complex essential for Escherichia coli survival? Nucleic Acids Res 44:4317-29|
|Chowdhury, Farhan; Li, Isaac T S; Ngo, Thuy T M et al. (2016) Defining Single Molecular Forces Required for Notch Activation Using Nano Yoyo. Nano Lett 16:3892-7|
|Kozlov, Alexander G; Weiland, Elizabeth; Mittal, Anuradha et al. (2015) Intrinsically disordered C-terminal tails of E. coli single-stranded DNA binding protein regulate cooperative binding to single-stranded DNA. J Mol Biol 427:763-74|
|Suksombat, Sukrit; Khafizov, Rustem; Kozlov, Alexander G et al. (2015) Structural dynamics of E. coli single-stranded DNA binding protein reveal DNA wrapping and unwrapping pathways. Elife 4:|
|Nguyen, Binh; Sokoloski, Joshua; Galletto, Roberto et al. (2014) Diffusion of human replication protein A along single-stranded DNA. J Mol Biol 426:3246-3261|
|Lee, Kyung Suk; Marciel, Amanda B; Kozlov, Alexander G et al. (2014) Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. J Mol Biol 426:2413-21|
|Antony, Edwin; Weiland, Elizabeth; Yuan, Quan et al. (2013) Multiple C-terminal tails within a single E. coli SSB homotetramer coordinate DNA replication and repair. J Mol Biol 425:4802-19|
|Kozlov, Alexander G; Galletto, Roberto; Lohman, Timothy M (2012) SSB-DNA binding monitored by fluorescence intensity and anisotropy. Methods Mol Biol 922:55-83|
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