Purification and characterization of the coliphage N4-coded single-stranded DNA binding protein.
We have purified and characterized a single-stranded DNA binding protein (N4 SSB) induced after coliphage N4 infection. It has a monomeric molecular weight of 31,000 and contains 10 tyrosine and 1-2 tryptophan amino acid residues. Its fluorescence spectrum is dominated by the tyrosine residues, and their fluorescence is quenched when the protein binds single-stranded DNA. Fluorescence quenching was used as an assay to quantitate binding of the protein to single-stranded nucleotides. The N4 single-stranded DNA binding protein binds cooperatively to single-stranded nucleic acids and binds single-stranded DNA more tightly than RNA. The binding involves displacement of cations from the DNA and anions from the protein. The apparent binding affinity is very salt-dependent, decreasing as much as 1,000-fold for a 10-fold increase in NaCl concentration. The degree of cooperativity (omega) is relatively independent of salt concentration. At 37 degrees C in 0.22 M NaCl, the protein has an intrinsic binding constant for M13 viral DNA of 3.8 x 10(4) M-1, a cooperativity factor omega of 300, and binding site size of 11 nucleotides per monomer. The protein lowers the melting point of poly(dA.dT).poly(dA-dT) by greater than 60 degrees C but cannot lower the melting transition or assist in the renaturation of natural DNA. N4 single-stranded DNA binding protein enhances the rate of DNA synthesis catalyzed by the N4 DNA polymerase by increasing the processivity of the N4 DNA polymerase and melting out hairpin structures that block polymerization.
Lindberg
G
G
Department of Biochemistry, University of Chicago, Illinois 60637.
Kowalczykowski
S C
SC
Rist
J K
JK
Sugino
A
A
Rothman-Denes
L B
LB
D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003090:Coliphages / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D007700:Kinetics; D008970:Molecular Weight; D009691:Nucleic Acid Denaturation; D011089:Polydeoxyribonucleotides; D013379:Substrate Specificity; D013698:Templates, Genetic
D004268:DNA-Binding Proteins; D011089:Polydeoxyribonucleotides