Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site

J.S Gibbs, Klaus Weisshart, Paul Digard, Anne DeBruynkops, David M Knipe, Donald M Coen

Research output: Contribution to journalArticlepeer-review

Abstract

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.
Original languageEnglish
Pages (from-to)4786-95
Number of pages10
JournalMolecular and Cellular Biology
Volume11
Issue number9
Publication statusPublished - Sep 1991

Keywords

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • Blotting, Southern
  • Blotting, Western
  • Cell Line
  • DNA Polymerase II/genetics
  • DNA Polymerase II/metabolism
  • DNA Replication
  • DNA, Bacterial
  • DNA, Recombinant
  • DNA, Viral
  • Escherichia coli/enzymology
  • Exodeoxyribonucleases/metabolism
  • Humans
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Osteosarcoma
  • Precipitin Tests
  • Sequence Homology, Nucleic Acid
  • Simplexvirus/genetics
  • Tumor Cells, Cultured

Fingerprint

Dive into the research topics of 'Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site'. Together they form a unique fingerprint.

Cite this