The Structure and Function of the Replication Initiator Protein (Rep) of pSC101: An Analysis Based on a Novel Positive-Selection System for the Replication-Deficient Mutants

J Biochem. 2001 Sep;130(3):399-405. doi: 10.1093/oxfordjournals.jbchem.a002999.

Abstract

Plasmid pSC101 encodes a 37.5 kDa Rep (RepA) protein, which binds to three 21-base repeats (DR-1, DR-2, and DR-3) in the replication origin region (ori) of the plasmid to initiate replication. Rep also binds to two palindromic sequences (IR-1 and IR-2) which overlap the rep promoter. The binding of Rep to IR-2 represses the production of Rep itself. It is highly likely that the balance of these functions of Rep plays a major role in controlling the copy number of pSC101. In this study, we developed a positive-selection system for replication-deficient mutants of the initiator protein. This system can be applied to the study of other replication systems by changing ori and rep of pSC101 to the corresponding genes. Thirty-four replication-deficient (Ini(-)) mutants were isolated with this system, and analyzed as to the relation between the structure and function of the Rep protein. Seventeen of these 34 Ini(-) mutants were found to lack auto-repressor activity as well as initiator activity. DNA sequence analysis showed that one-third (from the C-terminus) of Rep is dispensable for the auto-repressor activity, while the initiator activity seems to require the whole protein.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • DNA Helicases*
  • DNA Replication / genetics
  • DNA Replication / physiology
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Escherichia coli
  • Mutation, Missense / genetics*
  • Plasmids / genetics*
  • Promoter Regions, Genetic / genetics
  • Protein Structure, Tertiary / physiology
  • Proteins / genetics*
  • Proteins / metabolism
  • Repetitive Sequences, Nucleic Acid / genetics*
  • Repetitive Sequences, Nucleic Acid / physiology
  • Replication Origin / genetics*
  • Selection, Genetic
  • Trans-Activators*

Substances

  • Bacterial Proteins
  • DNA-Binding Proteins
  • Proteins
  • Trans-Activators
  • replication initiator protein
  • DNA Helicases