Participation of the fingers subdomain of Escherichia coli DNA polymerase I in the strand displacement synthesis of DNA.
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.
Singh
Kamalendra
K
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry-New Jersey Medical School, Newark, New Jersey 07103, USA. ksingh@umdnj.edu
Srivastava
Aashish
A
Patel
Smita S
SS
Modak
Mukund J
MJ
D020816:Amino Acid Motifs; D004247:DNA / Q000378:metabolism*; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000096:biosynthesis*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary / Q000235:genetics; D012313:RNA / Q000378:metabolism*
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C022262:Okazaki fragments; C034477:RNA primers; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D004256:DNA Polymerase I