A coupled complex of T4 DNA replication helicase (gp41) and polymerase (gp43) can perform rapid and processive DNA strand-displacement synthesis.
We have developed a coupled helicase-polymerase DNA unwinding assay and have used it to monitor the rate of double-stranded DNA unwinding catalyzed by the phage T4 DNA replication helicase (gp41). This procedure can be used to follow helicase activity in subpopulations in systems in which the unwinding-synthesis reaction is not synchronized on all the substrate-template molecules. We show that T4 replication helicase (gp41) and polymerase (gp43) can be assembled onto a loading site located near the end of a long double-stranded DNA template in the presence of a macro-molecular crowding agent, and that this coupled "two-protein" system can carry out ATP-dependent strand displacement DNA synthesis at physiological rates (400 to 500 bp per sec) and with high processivity in the absence of other T4 DNA replication proteins. These results suggest that a direct helicase-polymerase interaction may be central to fast and processive double-stranded DNA replication, and lead us to reconsider the roles of the other replication proteins in processivity control.
Dong
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F
Institute of Molecular Biology, University of Oregon, Eugene 97403-1129, USA.
Weitzel
S E
SE
von Hippel
P H
PH
D017122:Bacteriophage T4 / Q000235:genetics*; D001483:Base Sequence; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C063983:gene 41 protein, Enterobacteria phage T4; C069983:gene 43 protein, Enterobacteria phage T4; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases