Abstract
The replication of the genome requires the removal of RNA primers from the Okazaki fragments and their replacement by DNA. In prokaryotes, this process is completed by DNA polymerase I by means of strand displacement DNA synthesis and 5 '-nuclease activity. Here, we demonstrate that the strand displacement DNA synthesis is facilitated by the collective participation of Ser(769), Phe(771), and Arg(841) present in the fingers subdomain of DNA polymerase I. The steady and presteady state kinetic analysis of the properties of appropriate mutant enzymes suggest that: (a) Ser(769) and Phe(771) together are involved in the strand separation via the formation of a flap structure, and (b) Arg(841) interacts with the template strand to achieve the optimal strand separation and DNA synthesis. The amino acid residues Ser(769) and Phe(771) are constituents of the O1-helix, which together with O and O2 helices form a 3-helix bundle structure. We note that this 3-helix bundle motif also exists in prokaryotic RNA polymerase. Thus in both DNA and RNA polymerases, this motif may have been adopted to achieve the strand separation function.
Highlights
Strand displacement synthesis is an essential process in the removal and replacement of RNA primer moieties of Okazaki fragments
In the DNA-bound crystal structures of polymerase I (pol I) family DNA polymerases, the immediate unpaired template nucleotide assumes a flipped conformation [6] such that it cannot pair with the incoming dNTP substrate
The ternary complex crystal structure of KlenTaq shows that the 5Ј-phosphate group of the template nucleotide, which pairs with the incoming dNTP, interacts with Ser674 [6]
Summary
Materials—The PCR grade dNTPs were from Roche Applied Science. Radiolabeled nucleotides were obtained from PerkinElmer Life Sciences. The plasmid pCJ141 [17, 18] (provided by Dr Catherine Joyce of Yale University) was used for the generation of KF protein and to construct the desired mutant derivatives This plasmid contains the E. coli KF gene carrying a mutation, D424A. Reaction, the rates of single nucleotide incorporation were determined under single turnover conditions by rapid mixing of enzyme 32P-radiolabeled template primer blocker (32/ 14/14-mer; see Scheme 1) complex with Mg1⁄7dNTP, followed by quenching of the reaction by EDTA. Observed Catalytic Rates (kobs) of Single Nucleotide Incorporation of WT and Mutant Enzymes with Nicked DNA— The assays to determine the catalytic rates of single nucleotide incorporation were performed under the identical conditions mentioned above except for the DNA substrate, which was a nicked DNA consisting of a 28-mer template annealed with two 14-mer oligonucleotide (Scheme 1). Data Analysis—The amount of product formed (P) was graphed as a function of time (t), and the data were fit by nonlinear regression to the burst Equation 1,
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