Processivity of the gene 41 DNA helicase at the bacteriophage T4 DNA replication fork.
The gene 41 protein is the DNA helicase associated with the bacteriophage T4 DNA replication fork. This protein is a major component of the primosome, being essential for coordinated leading and lagging strand DNA synthesis. Models suggest that such DNA helicases are loaded only onto DNA at origins of replication, and that they remain with the ensuing replication fork until replication is terminated. To test this idea, we have measured the extent of processivity of the 41 protein in the context of an in vitro DNA replication system composed of eight purified proteins (the gene 43, 44/62, 45, 32, 41, 59, and 61 proteins). After starting DNA replication in the presence of these proteins, we diluted the 41 helicase enough to prevent any association of new helicase molecules and analyzed the replication products. We measured an association half-life of 11 min, revealing that the 41 protein is processive enough to finish replicating the entire 169-kilobase T4 genome at the observed replication rate of approximately 400 nucleotides/s. This processivity of the 41 protein does not require the 59 protein, the protein that catalyzes 41 protein assembly onto 32 protein-covered single-stranded DNA. The stability we measure for the 41 protein as part of the replication fork is greater than estimated for it alone on single-stranded DNA. We suggest that the 41 protein interacts with the polymerase holoenzyme at the fork, both stabilizing the other protein components and being stabilized thereby.
Schrock
R D
RD
Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California 94143-0448, USA.
Alberts
B
B
D017122:Bacteriophage T4 / Q000235:genetics*; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D014764:Viral Proteins / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C063983:gene 41 protein, Enterobacteria phage T4; C091225:gene 59 protein, Enterobacteria phage T4; D004265:DNA Helicases