RNA primer handoff in bacteriophage T4 DNA replication: the role of single-stranded DNA-binding protein and polymerase accessory proteins.
In T4 phage, coordinated leading and lagging strand DNA synthesis is carried out by an eight-protein complex termed the replisome. The control of lagging strand DNA synthesis depends on a highly dynamic replisome with several proteins entering and leaving during DNA replication. Here we examine the role of single-stranded binding protein (gp32) in the repetitive cycles of lagging strand synthesis. Removal of the protein-interacting domain of gp32 results in a reduction in the number of primers synthesized and in the efficiency of primer transfer to the polymerase. We find that the primase protein is moderately processive, and this processivity depends on the presence of full-length gp32 at the replication fork. Surprisingly, we find that an increase in the efficiency of primer transfer to the clamp protein correlates with a decrease in the dissociation rate of the primase from the replisome. These findings result in a revised model of lagging strand DNA synthesis where the primase remains as part of the replisome after each successful cycle of Okazaki fragment synthesis. A delay in primer transfer results in an increased probability of the primase dissociating from the replication fork. The interplay between gp32, primase, clamp, and clamp loader dictates the rate and efficiency of primer synthesis, polymerase recycling, and primer transfer to the polymerase.
Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Scott W
SW
Nelson
Ravindra
R
Kumar
Stephen J
SJ
Benkovic
D017122:Bacteriophage T4 / Q000201:enzymology / Q000235:genetics*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C034477:RNA primers; D014764:Viral Proteins; D012313:RNA; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase