Differential unstacking of nucleotides induced by binding of g5p and domain a of the human replication protein A.

摘要

The human replication protein A (hRPA) and gene 5 protein (g5p) encoded by the Ff strain Escherichia coli bacteriophage are single-stranded DNA-binding proteins (SSBs) involved in DNA metabolism. Both proteins exhibit OB-fold (oligonucleotide/oligosaccharide binding fold) motifs in their crystallographic structures, as well as having strong binding preference for pyrimidine-rich sequences. Though OB-fold SSBs are structurally similar, they generally lack sequence similarities except at their binding surfaces. This dissertation focuses on comparing nucleic acid perturbations caused by binding of two OB-fold proteins, g5p and RPA70A (major DNA-binding domain of hRPA). Techniques used to study the interactions of these SSBs with single-stranded DNAs are electrophoretic mobility shift assays (EMSAs), isothermal titration calorimetry (ITC), and circular dichroism (CD) and fluorescence spectroscopies. This dissertation reports the first use of ITC to derive thermodynamic parameters for the interactions of the two OB-fold proteins with ssDNAs and ultrafast time-resolved fluorescence spectroscopy has been explored as a means of studying nucleotide conformations bound at subsite loci within the DNA-binding channel of g5p.;Chapter 1 provides information on the crystallographic structure, binding modes, and biological functions of g5p and hRPA; details of techniques used to study g5p and RPA70A interactions with ssDNAs are also discussed in Chapter 1. Chapters 2 and 3 describe the innovative use of steady-state and time-resolved fluorescence spectroscopies to study the effect of g5p binding on 2-aminopurine (2AP) fluorescence labels located within single-stranded d(AC)8 tails of DNAs. Our studies revealed that regardless of the exact sequence context of the 2AP, binding of g5p significantly and differentially shifts its conformational equilibrium to an unstacked conformation at different subsite loci within the DNA-binding channel of g5p. In Chapter 4, steady-state fluorescence spectroscopy was used to monitor the perturbation of 2AP in a C-2AP-C sequence context within 5-nucleotide DNA oligomers (5-mers) bound by RPA70A. Nucleotides closest to the 5- and 3-ends of the 5-mer DNAs were minimally perturbed compared with the central nucleotide, which experienced the most significant unstacking upon RPA70A binding. ITC measurements showed that interactions of the OB-fold proteins with ssDNAs were enthalpically driven under comparable binding conditions. A summary of the thermodynamic parameters of these and related proteins is provided in Chapter 5, which concludes the dissertation and discusses future perspectives. Chapter 2 has been published in the American Chemical Society journal of Biochemistry.