Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding

Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these 'macromolecular machines'. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically i...

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Veröffentlicht in:Nucleic acids research 2015-10, Vol.43 (19), p.9276-9290
Hauptverfasser: Jose, Davis, Weitzel, Steven E, Baase, Walter A, von Hippel, Peter H
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container_end_page 9290
container_issue 19
container_start_page 9276
container_title Nucleic acids research
container_volume 43
creator Jose, Davis
Weitzel, Steven E
Baase, Walter A
von Hippel, Peter H
description Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these 'macromolecular machines'. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically introduced base analogues to map and quantify the equilibrium binding interactions of short (8 nts) ssDNA oligomers with gp32 monomers at single nucleotide resolution. We show that single gp32 molecules interact most directly and specifically near the 3'-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2-3 nts are directly involved in this tight binding interaction. The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3'-end and facilitates base unstacking along the entire 8-mer lattice. These results provide a model (and 'DNA map') for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex.
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subjects 2-Aminopurine
Bacteria
Binding Sites
Circular Dichroism
DNA, Single-Stranded - chemistry
DNA, Single-Stranded - metabolism
DNA-Binding Proteins - chemistry
DNA-Binding Proteins - metabolism
Genome Integrity, Repair and
Nucleotides - chemistry
Protein Binding
Protein Multimerization
Viral Proteins - chemistry
Viral Proteins - metabolism
title Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding
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