Mapping DNA conformations and interactions within the binding cleft of bacteriophage T4 single-stranded DNA binding protein (gp32) at single nucleotide resolution
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Date
2020-12-24
Authors
Camel, Benjamin R.
Jose, Davis
Meze, Katarina
Dang, Anson
von Hippel, Peter H.
Journal Title
Journal ISSN
Volume Title
Publisher
Oxford University Press
Abstract
In this study, we use single-stranded DNA (oligo-dT) lattices that have been position-specifically labeled with monomer or dimer 2-aminopurine (2-AP) probes to map the local interactions of the DNA bases with the nucleic acid binding cleft of gp32, the single-stranded binding (ssb) protein of bacteriophage T4. Three complementary spectroscopic approaches are used to characterize these local interactions of the probes with nearby nucleotide bases and amino acid residues at varying levels of effective protein binding cooperativity, as manipulated by changing lattice length. These include: (i) examining local quenching and enhancing effects on the fluorescence spectra of monomer 2-AP probes at each position within the cleft; (ii) using acrylamide as a dynamic-quenching additive to measure solvent access to monomer 2-AP probes at each ssDNA position; and (iii) employing circular dichroism spectra to characterize changes in exciton coupling within 2-AP dimer probes at specific ssDNA positions within the protein cleft. The results are interpreted in part by what we know about the topology of the binding cleft from crystallographic studies of the DNA binding domain of gp32 and provide additional insights into how gp32 can manipulate the ssDNA chain at various steps of DNA replication and other processes of genome expression.
Description
12 pages
Genome integrity
Genome repair
Genome replication
Genome integrity
Genome repair
Genome replication
Keywords
Citation
Benjamin R Camel, Davis Jose, Katarina Meze, Anson Dang, Peter H von Hippel, Mapping DNA conformations and interactions within the binding cleft of bacteriophage T4 single-stranded DNA binding protein (gp32) at single nucleotide resolution, Nucleic Acids Research, Volume 49, Issue 2, 25 January 2021, Pages 916–927, https://doi.org/10.1093/nar/gkaa1230