Identification of conserved residues contributing to the activities of the adenovirus DNA polymerase

H Liu, James Henderson Naismith, Ronald Thomas Hay

Research output: Contribution to journalArticlepeer-review

22 Citations (Scopus)

Abstract

Adenovirus codes for a DNA polymerase that is a member of the DNA polymerase a family and uses a protein primer for initiation of DNA synthesis. It contains motifs characteristic of a proofreading 3 ' -5 ' -exonuclease domain located in the N-terminal region and several polymerase motifs located in the C-terminal region. To determine the role of adenovirus DNA polymerase in DNA replication, 22 site-directed mutations were introduced into the conserved DNA polymerase motifs in the C-terminal region of adenovirus DNA polymerase and the mutant forms were expressed in insect cells using a baculovirus expression system. Each mutant enzyme was tested for DNA binding activity, the ability to interact with pTP, DNA polymerase catalytic activity, and the ability to participate in the initiation of adenovirus DNA replication. The mutant phenotypes identify functional domains within the adenovirus DNA polymerase and allow discrimination between the roles of conserved residues in the various activities carried out by the protein. Using the functional data in this study and the previously published structure of the bacteriophage RB69 DNA polymerase (J. Wang et al., Cell 89:1087-1099, 1997), it is possible to envisage how the conserved domains in the adenovirus DNA polymerase function.

Original languageEnglish
Pages (from-to)11681-11689.
Number of pages9
JournalJournal of Virology
Volume74
Issue number24
DOIs
Publication statusPublished - Dec 2000

Keywords

  • LINKER-INSERTION MUTAGENESIS
  • CRYSTAL-STRUCTURE
  • TERMINAL PROTEIN
  • ANGSTROM RESOLUTION
  • PRETERMINAL PROTEIN
  • LARGE FRAGMENT
  • ACTIVE-SITE
  • DOMAIN ORGANIZATION
  • MUTATIONAL ANALYSIS
  • TEMPLATE-PRIMER

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