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J. Biol. Chem., Vol. 278, Issue 28, 25435-25447, July 11, 2003
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From the Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403
Complexes formed between DNA polymerase and genomic DNA at the replication
fork are key elements of the replication machinery. We used sedimentation
velocity, fluorescence anisotropy, and surface plasmon resonance to measure
the binding interactions between bacteriophage T4 DNA polymerase (gp43) and
various model DNA constructs. These results provide quantitative insight into
how this replication polymerase performs template-directed 5' 
3' DNA synthesis and how this function is coordinated with the
activities of the other proteins of the replication complex. We find that
short (single- and double-stranded) DNA molecules bind a single gp43
polymerase in a nonspecific (overlap) binding mode with moderate affinity
(Kd 
150 nM) and a binding site
size of 10 nucleotides for single-stranded DNA and 13 bp for
double-stranded DNA. In contrast, gp43 binds in a site-specific (nonoverlap)
mode and significantly more tightly (Kd 5
nM) to DNA constructs carrying a primer-template junction, with the
polymerase covering 5 nucleotides downstream and 6–7 bp
upstream of the 3'-primer terminus. The rate of this specific binding
interaction is close to diffusion-controlled. The affinity of gp43 for the
primer-template junction is modulated specifically by dNTP substrates, with
the next "correct" dNTP strengthening the interaction and an
incorrect dNTP weakening the observed binding. These results are discussed in
terms of the individual steps of the polymerase-catalyzed single nucleotide
addition cycle and the replication complex assembly process. We suggest that
changes in the kinetics and thermodynamics of these steps by auxiliary
replication proteins constitute a basic mechanism for protein coupling within
the replication complex.
Received for publication, April 1, 2003 , and in revised form, April 11, 2003.
* This work was supported in part by U.S. Public Health Service Research Grants GM15792 and GM29158. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
An American Cancer Society Research Professor of Chemistry. To whom
correspondence should be addressed. Tel.: 541-346-6097; Fax: 541-346-5891;
E-mail:
petevh{at}molbio.uoregon.edu.
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