Laser cross-linking of proteins to nucleic acids. II. Interactions of the bacteriophage T4 DNA replication polymerase accessory proteins complex with DNA.
In this paper we examine the interactions of the polymerase accessory proteins subassembly of the bacteriophage T4 DNA replication complex, using single-pulse ultraviolet laser excitation to induce protein-nucleic acid cross-links. The laser-induced cross-linking permits effective "freezing" of the instantaneous equilibrium state of the complex and thus provides a mechanism to dissect the individual protein-nucleic acid interactions involved in complex assembly. We find that the binding of the gene 44, 62, and 45 proteins is dependent not only on the presence of each of the other proteins, but also on the presence of adenine nucleotide cofactors. We find that the nonhydrolyzable analogs of ATP often behave more like ADP than ATP in these experiments. Gene 45 protein is able to induce an increase in cross-linking of the gp44/62 complex to nucleic acids, and this increased cross-linking correlates with changes in the apparent Km of the gp44/62 complex for polynucleotides and with changes in Vmax during ATP hydrolysis. Our results suggest that the enhanced DNA binding is predominately through the gene 62 protein and not the ATPase catalytic subunit (gene 44 protein). Thus the gene 62 protein seems to play an integral role in gp45-mediated enhancement of the ATP hydrolytic activity of gp44. These results are summarized and integrated in the form of a model for the multiple interactions of the accessory proteins with DNA and one another in the presence of mononucleotide cofactors and substrates.
Hockensmith
J W
JW
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Kubasek
W L
WL
Evertsz
E M
EM
Mesner
L D
LD
von Hippel
P H
PH
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D004261:DNA Replication; D004279:DNA, Viral / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D007834:Lasers; D013604:T-Phages / Q000737:chemistry*; D013698:Templates, Genetic; D014764:Viral Proteins / Q000737:chemistry*
D004279:DNA, Viral; D014764:Viral Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004259:DNA-Directed DNA Polymerase