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Probing the nucleic acid binding modes of the single‐stranded DNA binding protein of the bacteriophage T4 replication complex
Author(s) -
Jose Davis,
Weitzel Steven E.,
Hippel Peter H.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.541.2
Subject(s) - oligonucleotide , biophysics , dna , nucleic acid , replisome , binding site , bacteriophage , biology , circular dichroism , dna replication , oligomer , macromolecule , chemistry , microbiology and biotechnology , biochemistry , gene , escherichia coli , eukaryotic dna replication , organic chemistry
Combining biophysical measurements on the function and control of T4 bacteriophage replication complexes with detailed structural information can throw light onto the mechanism of action of these â€̃macromolecular machines’. In this study we use the low energy circular dichroism and fluorescent properties of site‐specifically introduced fluorescent base analogues to monitor the binding of gene 32 protein (gp32), the ssDNA binding protein of the T4 DNA replication complex, to ssDNA oligomers ranging from 8 to 60 nts in length. We show that gp32 binds preferentially to the 3’‐end of a short ssDNA oligomer and that only two or three nts at the 3’ end are directly involved in the binding interaction. Loss of exciton coupling in the CD spectrum and an increase in fluorescence intensity shows that gp32 binding causes extension of sugar‐phosphate backbone of the oligonucleotide. Further, our results suggest that sliding (or â€̃shuffling’) of the oligonucleotide along the nucleic acid binding cleft of the protein is not observed in the isolated single‐site gp32 DNA binding mode. Mechanisms by which gp32 binds to and covers its ssDNA targets in both an isolated and a cooperative binding mode will be discussed.