Mutational analysis of the specific priming signal essential for DNA replication of the broad host-range plasmid RSF1010

FEBS Lett. 1993 Jun 7;324(1):67-70. doi: 10.1016/0014-5793(93)81534-7.

Abstract

To analyze the RSF1010-specific priming mechanism, a library of randomly mutagenized ssiA sequences was constructed by chemical synthesis using mixed nucleotide phosphoramidites. Synthetic ssiA sequences with the single base-substitutions were assayed for the SSI activity in E. coli JM109 expressing RepB' primase. It was demonstrated that the activity of ssiA was damaged markedly by single base-substitutions within the possible stem-loop structure and its 3'-flanking region. It is conceivable that these domains are critical in recognition and primer synthesis by RepB' primase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Composition
  • Base Sequence
  • DNA Replication / genetics*
  • DNA, Bacterial / genetics
  • DNA, Single-Stranded / genetics*
  • Escherichia coli / genetics*
  • Gene Library
  • Genes, Synthetic
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nucleic Acid Conformation
  • Oligodeoxyribonucleotides
  • Plasmids*
  • Restriction Mapping

Substances

  • DNA, Bacterial
  • DNA, Single-Stranded
  • Oligodeoxyribonucleotides