engleski

Structural characterization of Streptomyces coelicolor single-stranded DNA-binding proteins and their complexes

Single-stranded DNA-binding proteins (SSBs) are required in DNA replication, recombination and repair, in all organisms, from bacteria to human. Their ability to bind single-stranded DNA (ssDNA) with high affinity makes them important for protection of ssDNA from chemical and nuclease attacks and from forming aberrant secondary structures. Bacterial SSBs have two distinct domains: N-terminal domain responsible for DNA binding and C-terminal tail that mediates protein-protein interactions (1, 2). Bacteria Streptomyces coelicolor encodes two SSBs (ScSSB) that differ in size: larger contains 199 amino acids (3) and shorter 156 amino acids (data not published). Recently, the crystal structure of larger ScSSB has been solved (data not published), and first crystals of short ScSSB have been obtained. Because of low diffraction quality of the crystals, new crystallization conditions should be found. Our goal is also to prepare ScSSB-ssDNA complexes, crystallize them and solve their structures. Currently, we are trying to characterize SSB-ssDNA complex in order to determine binding properties (intrinsic binding constant (K), nucleotide binding site-size (n) and cooperativity parameter (ω )). Quenching of the intrinsic tryptophan fluorescence of the SSB by the nucleic acid is used to monitor binding. This characterization will help in generating homogeneus complexes necessary for preparing crystals for structure determination. References: 1. Lohman, T.M., Ferrari, M.E. (1994) Escherichia coli single-stranded DNA-binding protein: Multiple DNA binding modes and cooperativities. Annu. Rev. Biochem., 63:527-570. 2. Raghunathan, S., Kozlov, A.G., Lohman, T.M., Waksman, G. (2000) Structure of the DNA binding domain of E. coli SSB bound to ssDNA. Nature Struct. Biol., 7:648-652. 3. Mijakovic, I., Petranovic, D., Macek, B., Cepo, T., Mann, M., Davies, J., Jensen, P.R., Vujaklija, D. (2006) Bacterial singlestranded DNA-binding proteins are phosphorylated on tyrosine. Nucleic Acids Res., 34: 1588-1596

single-stranded DNA-binding protein; protein-DNA interactions; Streptomyces coelicolor

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