Protein-DNA cross-linking demonstrates stepwise ATP-dependent assembly of T4 DNA polymerase and its accessory proteins on the primer-template.
T4 DNA polymerase, the 44/62 and 45 polymerase accessory proteins, and 32 single-stranded DNA-binding protein catalyze ATP-dependent DNA synthesis. Using DNA primers with cross-linkable residues at specific positions, we obtained structural data that reveal how these proteins assemble on the primer-template. With the nonhydrolyzable ATP analog ATP gamma S, assembly of the 44/62 and 45 proteins on the primer requires 32 protein but not polymerase. ATP hydrolysis changes the position and intensity of cross-linking to each of the accessory proteins and allows cross-linking of polymerase. Our data indicate that the initial binding of the three accessory proteins and ATP to a 32 protein-covered primer-template is followed by ATP hydrolysis, binding of polymerase, and movement of the accessory proteins to yield a complex capable of processive DNA synthesis.
Capson
T L
TL
Department of Chemistry, Pennsylvania State University, University Park 16802.
Benkovic
S J
SJ
Nossal
N G
NG
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D003432:Cross-Linking Reagents; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D013698:Templates, Genetic
D003432:Cross-Linking Reagents; D004279:DNA, Viral; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases