Biomedical subjects

A J Spiers

Publications and source records attributed to A J Spiers.

6 recordsLinked to original sources

PubMed1999

C-terminal interactions between the XerC and XerD site-specific recombinases.

Studies of the site-specific recombinase Cre suggest a key role for interactions between the C-terminus of the protein and a region located about 30 residues from the C-terminus in linking in a cyclical manner the four recombinase monomers present in a recombination complex, and in controlling the catalytic activity of each monomer. By extrapolating the Cre DNA recombinase structure to the related site-specific recombinases XerC and XerD, it is predicted that the extreme C-termini of XerC and XerD interact with alpha-helix M in XerD and the equivalent region of XerC respectively. Consequently, XerC and XerD recombinases deleted for C-terminal residues, and mutated XerD proteins containing single amino acid substitutions in alphaM or in the C-terminal residues were analysed. Deletion of C-terminal residues of XerD has no measurable effect on co-operative interactions with XerC in DNA-binding assays to the recombination site dif, whereas deletion of 5 or 10 residues of XerC reduces co-operativity with XerD some 20-fold. Co-operative interactions between pairs of truncated proteins during dif DNA binding are reduced 20- to 30-fold. All of the XerD mutants, except one, were catalytically proficient in vitro; nevertheless, many failed to mediate a recombination reaction on supercoiled plasmid in vivo or in vitro, implying that the ability to form a productive recombination complex and/or mediate a controlled recombination reaction is impaired.

Amino Acid Sequence↗

PubMed1997

Relating primary structure to function in the Escherichia coli XerD site-specific recombinase.

XerC and XerD are related 298-amino-acid site-specific recombinases, each of which is responsible for the exchange of one pair of strands in Xer recombination. Both recombinases encode functions necessary for sequence-specific DNA-binding, co-operative XerC/D interactions, synapsis and catalysis. These functions were related to the primary amino acid sequence by constructing and analysing internal and C-terminal XerD deletions. An XerD derivative containing residues 1-233 was proficient in specific DNA binding, but did not interact co-operatively with XerC. Deletion of a further five C-terminal amino acids abolished binding to DNA. Proteins deleted for residues 32-88 and for residues 145-159 were deficient in DNA binding. Deletion of residues 244-281, a region containing amino acids necessary for catalysis, gave a protein that bound to DNA. An XerD derivative containing residues 1-268 retained co-operative interactions with XerC; nevertheless, it did not support XerC strand exchange and was defective in XerD catalysis. Residues 1-283 retain a functional catalytic active site, though a protein lacking the five C-terminal amino acids was still unable to mediate normal in vivo recombination, indicating that these residues are needed for a function that is not directly related to DNA binding or catalysis.

Amino Acid Sequence↗

PubMed1993

RepFIB: a basic replicon of large plasmids.

The distribution of the RepFIB replicon among a total of 20 plasmid incompatibility groups was determined using Southern blot and polymerase chain reaction analysis. The presence of the replicon was confirmed in 25 of 55 plasmids tested. The majority of plasmids carrying RepFIB are from the IncF incompatibility groups, but one plasmid from IncI and one plasmid from IncP also possess the replicon. Seven different examples of RepFIB were sequenced in the minimal replicon region to obtain 1525 bp of sequence information covering the repA gene and flanking repeat regions for comparison. An analysis of these sequences plus three sequences previously reported showed almost perfect conservation of the predicted protein sequence of RepA and of the flanking DNA repeats. DNA sequence data were analyzed using maximum parsimony techniques to describe the possible evolutionary relationships of the 10 examples of RepFIB.

Amino Acid Sequence↗

PubMed1993

Regulatory interactions between RepA, an essential replication protein, and the DNA repeats of RepFIB from plasmid P307.

The control of RepFIB replication appears to rely on the interaction between an initiator protein (RepA) and two sets of DNA repeat elements located on either side of the repA gene (BCDD'D" and EFGHIJ). In vivo genetic tests demonstrate that the BCDD'D" repeats form part of the origin of replication, while some of the downstream repeat elements (HIJ) are involved in the sensing and setting of plasmid copy number. RepA DNA binding to these groups of repeats has been investigated in vivo by utilizing the fact that the replicon contains three active promoters (orip, repAp, and EFp), one of which has previously been shown to control the expression of repA (repAp). All three promoters are closely associated with the repeat elements flanking repA, and an investigation using lacZ or cml gene fusions has demonstrated that RepA expressed in trans is able to repress each promoter. However, these assays suggest that the transcriptional responses of orip and repAp to RepA repression are significantly different, despite the fact that both promoters are embedded within the BCDD'D" repeat elements. Extra copies of the BCDD'D" or EFG repeats in trans have no effect on RepA repression of repAp embedded in a second copy of the BCDD'D" repeats, but copies of the HIJ or EFGHIJ repeats are able to derepress repAp, suggesting that there is a fundamental difference between RepA-BCDD'D" or -HIJ complexes and RepA-EFG or -EFGHIJ complexes.

Bacterial Proteins↗

PubMed1992

Expression and regulation of the RepA protein of the RepFIB replicon from plasmid P307.

The control of RepFIB replication appears to rely on the interaction between an initiator protein (RepA) and two sets of DNA repeat elements located on either side of the repA gene. Limited N-terminal sequence information obtained from a RepA:beta-galactosidase fusion protein indicates that although the first residue of RepA is methionine, the initiation of translation of RepA occurs from a CTG codon rather than from the predicted GTG codon located further downstream. Overexpressed RepA in trans is capable of repressing a repA:lacZ fusion plasmid in which the expression of the fusion protein is under the control of the repA promoter. The repA promoter has been located functionally by testing a series of repA:lacZ fusion plasmids. Both in vivo genetic tests and in vitro DNA-binding studies indicate that repA autoregulation can be achieved by RepA binding to one or more repeat elements which overlap the repA promoter sequence.

Bacterial Proteins↗

PubMed1989

Nucleotide sequence and replication characteristics of RepFIB, a basic replicon of IncF plasmids.

A second autonomous replicon of P307, RepFIB, has been isolated that has significant homology with other replicons in IncFI group plasmids. Eleven homologous repeats of 21 base pairs are present on the sequence and flank an open reading frame capable of coding for a protein of about Mr = 40,000. This protein was identified by maxicell analysis of cloned RepFIB. A series of deletion mutations of RepFIB were inserted into a DNA polymerase I-dependent vector and examined for their replication proficiency in a polA1 strain. These experiments defined a minimal replication region of 1.6 kilobases which includes the three repeats immediately upstream and downstream of the open reading frame. Deletion of a second set of repeats further downstream doubled the copy number of a chimeric plasmid replicating under RepFIB control. It was concluded that these repeats control the copy number of the replicon. Incompatibility tests showed that all three sets of repeats could express incompatibility with a resident RepFIB plasmid.

Base Sequence↗