engleski

Thermodynamic insight into binding properties of paralogous SSB proteins of Streptomyces coelicolor with ssDNA

Streptomyces species exhibit a complex and tightly regulated developmental life cycle. Cell proliferation depends primarily on accurate chromosome replication. In all domains of life single-strand DNA-binding (SSB) proteins have a key role in DNA metabolism. SSBs bind to single- stranded DNA (ssDNA) with high affinity but low sequence specificity. They bind transiently single-stranded DNA (ssDNA) formed during various cellular events and protect it from nucleolytic degradation. In addition, these proteins also interact with different cellular proteins, modulate their activities, and orchestrate DNA replication, repair, and recombination. Various bacteria possess paralogous SSBs, but their biological role is poorly understood. SSB has an N-terminal domain which is responsible for ssDNA binding and the C-terminal domain. The last one contains an acidic tip and an intrinsically disordered C-terminal linker (IDL) rich in glycine residues. This flexible domain ends with a specific acidic tip (8-10 aa) which is highly conserved in many bacterial SSB proteins. Recent studies have shown that the removal of the acidic tip results in the elimination of binding to the target proteins and also affects cooperative binding to ssDNA as well as the stability of the SSB tetramer. However, contemporary studies showed that high cooperative binding of model SSB protein (SSB) to ssDNA in its (SSB)35 or (SSB)65/(SSB)56 modes require IDL region. Within the IDL region, it has been shown the presence of repetitive elements containing G, P, and Q, a putative polyproline type helix (PPII), and a PXXP motif are important for SSB function. Based on these results IDL was proposed as a crucial region for protein-protein interaction and the PXXP motif as the binding site to the OB-fold of either another SSB tetramer or another partner protein. The objectives of this study were to explore the binding affinity of the two S. coelicolor paralogous SSB proteins (SsbA and SsbB) to ssDNA. It is known that they come in a form of homotetramers that can bind ssDNA in multiple binding modes ; (SSB)35 and (SSB)65. Various genetic constructs were prepared to analyze how deletion of the above-described elements influence the affinity binding of these proteins with oligonucleotides using isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy.

Thermodynamic ; Proteins ; Motif ; Binding

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