Primary structure of the replication initiation protein of plasmid R6K.

Abstract

The cistron of the replication initiation protein of plasmid R6K has been cloned into the single-strand DNA vectors M13mp8 and M13mp9 and its complete nucleotide sequence has been determined. The amino acid sequence of the initiator protein as predicted from its nucleotide sequence shows that the protein is lysine rich and weakly basic and has a molecular weight of 35,000, which is in close agreement with that estimated from the mobility in NaDodSO4/acrylamide gels. The secondary structure of the protein, approximated by the probabilistic methods of Chou and Fasman [Chou, P. & Fasman, G. (1978) Adv. EnzymoL 47, 45-148], suggests an NH2-terminal domain of primarily positively charged a-helical structure, a core region of interspersed short stretches of random coils and a8-sheets and -turns, and a COOH-terminal domain of a-helix.The mechanism of the initiation of replication of doublestranded DNA molecules that replicate in a topologically symmetric configuration (i.e., both strands of the DNA are replicated by similar or identical mechanisms), from a specific origin is a major unsolved problem in molecular biology. A class of proteins called initiator proteins are involved in the potentiation ofthe first replication forks at the origin. A detailed understanding of the structure, enzymatic properties, and site of action (on the DNA) of this class ofproteins is essential for unraveling the molecular mechanism of initiation of replication.The drug-resistance factor R6K, which confers resistance to ampicillin and streptomycin and has a molecular weight of 26 million (1), replicates in a Cairns type configuration from multiple origins of replication (2,3). An in vitro system for replicating R6K DNA and its derivatives is available (4, 5); the plasmid encodes its own initiator protein (5, 6) and contains a specific replication terminus (7,8). These characteristics recommend the plasmid as a convenient system to study the mechanisms of initiation and termination of replication.The initiation protein of R6K has been shown to be encoded in the DNA sequence contained in the HindIII fragments 9 and 15 (5, 6). Subcloning experiments had established that a combination of the restriction fragments HindIII 9-15-2 and 15-9-4 yielded self-replicating miniplasmids (5) having the replication origins a and y (2, 3) located in the HindIII fragment 4 and the replication origin ,3 located in the HindIII fragment 2 (refs. 2, 6; unpublished work).To study the molecular biology of the initiation ofreplication in R6K, we have cloned the cistron of this protein in the singlestrand phage vectors M13mp5, M13mp8, and M13mp9, and in this report we present the complete nucleotide sequence of the cistron and the amino acid sequence of the initiator protein as predicted from its nucleotide sequence. MATERIALS AND METHODSBacterial Strains, Phage Strains, and Plasmids. Escherichia coli strain JM 103 (Mlac, pro-i, SupE, thi, endA, sbcB15, hsdR4, lacd, lacZml3, F' proA , proB+) and the M13 strains mp8 and mp9 were obtained from J. Messing th...

Completion of the Sequence of the Genome of the Coronavirus Avian Infectious Bronchitis Virus

“…One of the most interesting matches is at the 5' end of the first large ORF. The first 300 amino acids have a low-level but extensive homology with the replication initiation protein from Escherichia coli (Germino & Bastia, 1982). The homology is statistically significant and it may indicate that this region of the polymerase protein is involved in initiation of replication of either the positive or negative strands.…”

Crystal structure of π initiator protein–iteron complex of plasmid R6K: Implications for initiation of plasmid DNA replication

“…In fact, the structure of only one plasmidencoded initiator, namely RepE of F factor, is known to date (13). Unfortunately, the F plasmid system offers very few mutants of the RepE initiator that have been isolated and analyzed to date and very little published information exists on the interaction of the RepE initiator with host-encoded replication proteins.By contrast, the R6K plasmid system has a rich repertoire of biological and biochemical information on its initiator protein called (2,3,6,8,(14)(15)(16)(17)(18)(19), including its interaction with several host-encoded replication proteins (20-22), and its self interaction, via dimerization (23, 24), dimer-dimer (or dimer-monomer) interactions, that are essential for DNA looping (22). The R6K replicons consist of three replication origins called ␣, ␤, and ␥.…”

“…I n a major class of drug resistance plasmids, interaction of a plasmid-encoded replication initiator protein with the originproximal, cognate, repeated sequences called iterons, initiates Cairns-type replication that generates -shaped replication intermediates (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Although a wealth of information exists on the genetics and biochemistry of replication initiation and its control in a few model plasmid systems, there has been a paucity of information on the atomic structure of the plasmid-encoded replication initiator proteins and their detailed structurefunction analysis.…”

“…By contrast, the R6K plasmid system has a rich repertoire of biological and biochemical information on its initiator protein called (2,3,6,8,(14)(15)(16)(17)(18)(19), including its interaction with several host-encoded replication proteins (20)(21)(22), and its self interaction, via dimerization (23,24), dimer-dimer (or dimer-monomer) interactions, that are essential for DNA looping (22). The R6K replicons consist of three replication origins called ␣, ␤, and ␥.…”

Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein

“…All three origins require the essential 277-bp core segment, which contains repeated binding sites (iterons) for TT (14,19,33); each origin also needs a unique cis-acting DNA segment in addition to the core (28,29,36,41,42,44). When the flanking ot and P sequences are removed, the remaining y origin can replicate autonomously (18,43,46) if thepir gene is provided in cis or in trans (22,29). The y origin responds to the intracellular concentration of uz protein, and it contains the most critical information required for replication (12,28).…”