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 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.
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403-1229, USA.
Davis
D
Jose
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403-1229, USA.
Steven E
SE
Weitzel
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403-1229, USA.
Walter A
WA
Baase
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403-1229, USA petevh@molbio.uoregon.edu.
Peter H
PH
von Hippel
D015075:2-Aminopurine; D001665:Binding Sites; D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D055503:Protein Multimerization; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009711:Nucleotides; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D015075:2-Aminopurine