xerB, an Escherichia coli gene required for plasmid ColE1 site-specific recombination, is identical to pepA, encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase.

Stirling, Colin J.; Colloms, Sean D.; Collins, John; Szatmari, George; Sherratt, David J.

doi:10.1002/j.1460-2075.1989.tb03547.x

Cited by 173 publications

(123 citation statements)

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“…For example, the E. coli LAP, also known as XerB, PepA and CarP, appears to be multifunctional. The E. coli LAP serves as an aminopeptidase (Vogt, 1970) and a DNA-binding protein that mediates both site-specific recombination at the cer site of ColE1 plasmids (Stirling et al, 1989) and transcriptional activation of the carAB operon (Charlier et al, 2000). The DNAbinding capabilities of the E. coli LAP are independent of aminopeptidase function (McCulloch et al, 1994;Charlier et al, 2000).…”

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Isolation and Characterization of the Neutral Leucine Aminopeptidase (LapN) of Tomato

Park

Walling

2003

Plant Physiology

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Tomatoes (Lycopersicon esculentum) express two forms of leucine aminopeptidase (LAP-A and LAP-N) and two LAP-like proteins. The relatedness of LAP-N and LAP-A was determined using affinity-purified antibodies to four LAP-A protein domains. Antibodies to epitopes in the most N-terminal region were able to discriminate between LAP-A and LAP-N, whereas antibodies recognizing central and COOH-terminal regions recognized both LAP polypeptides. Two-dimensional immunoblots showed that LAP-N and the LAP-like proteins were detected in all vegetative (leaves, stems, roots, and cotyledons) and reproductive (pistils, sepals, petals, stamens, and floral buds) organs examined, whereas LAP-A exhibited a distinct expression program. LapN was a single-copy gene encoding a rare-class transcript. A full-length LapN cDNA clone was isolated, and the deduced sequence had 77% peptide sequence identity with the wound-induced LAP-A. Leucyl aminopeptidases (LAPs; EC 3.4.11.1) are members of the M17 family of peptidases (Barrett et al., 1998). LAPs are ubiquitous being found in animals, plants, and prokaryotic cells. These hexameric metallopeptidases catalyze the release of the N-terminal residues from protein, peptide, fluorometric, and chromogenic substrates. The best characterized LAPs are from Bos taurus, Escherichia coli and tomato (Lycopersicon esculentum). X-ray crystal structures of the bovine and E. coli LAPs have provided insight into the LAP catalytic mechanism (Kim and Lipscomb, 1994;Sträter and Lipscomb, 1995;Sträter et al., 1999a). The roles of selected residues of the E. coli and tomato LAPs in chromogenic or peptide substrate catalysis, respectively, have been tested by site-directed mutagenesis (Sträter et al., 1999b;Gu and Walling, 2002).In most plants, three classes of LAP-related polypeptides are detected using a tomato LAP antiserum, including the 66-and 77-kD LAP-like proteins and the 55-kD neutral LAP (LAP-N; Chao et al., 2000). Only in a subset of the Solanaceae is a second 55-kD LAP species (LAP-A) detected (Hildmann et al., 1992;Gu et al., 1996b;Chao et al., 2000). In tomato, LAP-A protomers have an acidic pI and are encoded by two genes (LapA1 and LapA2), which are expressed during floral and fruit development. The LapA genes are not expressed in foliage from healthy plants (Chao et al., 1999). However, LapA RNAs, proteins, and activities accumulate locally and systemically in leaves after wounding, Pseudomonas syringae pv. tomato and Phytophthora parasitica infection, and caterpillar feeding (Pautot et al., 1993(Pautot et al., , 2001Gu et al., 1996b;Chao et al., 1999;Jwa and Walling, 2001). The activation of LapA gene expression by jasmonic acid (JA), abscisic acid, the phytotoxin coronatine (a JA mimic), and suppression of LapA by salicylic acid is consistent with the regulation of the tomato LapA genes by the wound octadecanoid pathway (Chao et al., 1999). LapA genes also respond to signals generated during water deficit and salinity stress (Chao et al., 1999). The potato (Solanum tuberosum) Lap RNAs also ac...

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Isolation and Characterization of the Neutral Leucine Aminopeptidase (LapN) of Tomato

Park

Walling

2003

Plant Physiology

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“…Protease-deficient E. coli CM 89 [leu-9 A(pro lac) met thyA pepN102 pepAll pepB1 pepQ10] and CM 17 [leu-9 A(pro lac) met thyA] (18) were received from Charles G. Miller. E. coli DS947 xerBl A(lac pro), the 2-cer reporter plasmid pCS210, and pCS126 containing the E. coli xerB gene (24) were received from David J. Sherratt. E. coli strains were cultured at 370C in LB medium (9).…”

Section: Methodsmentioning

confidence: 99%

“…Bovine lens aminopeptidase, a zinc metalloenzyme, consists of six identical subunits each with a molecular mass of 51,691 Da (6,8). The amino acid sequence of the bovine protein has been obtained (8), and recently, bacterial (xerB) and plant gene analogs have been identified (3,24). Using peptidase mutants of Salmonella typhimurium, Stirling and coworkers (24) concluded that xerB represents the Escherichia coli analog of the pepA gene of S. typhimurium that codes for a peptidase with broad specificity and known involvement in the pathway of protein degradation (15,16).…”

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“…The amino acid sequence of the bovine protein has been obtained (8), and recently, bacterial (xerB) and plant gene analogs have been identified (3,24). Using peptidase mutants of Salmonella typhimurium, Stirling and coworkers (24) concluded that xerB represents the Escherichia coli analog of the pepA gene of S. typhimurium that codes for a peptidase with broad specificity and known involvement in the pathway of protein degradation (15,16). Interestingly, E. coli XerB/PepA not only possesses peptidase activity but is also involved in a site-specific recombination system that recognizes plasmid cer sites and resolves unstable ColEl and related plasmid multimers into their monomeric forms (24 During a screening with oligonucleotide probes targeting genes coding for rickettsial tricarboxylic acid cycle enzymes (34), a recombinant that in initial sequence analysis exhibited similarity to both procaryotic and eucaryotic leucine aminopeptidase was identified.…”

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Characterization of the Rickettsia prowazekii pepA gene encoding leucine aminopeptidase

1993

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PepA with the characterized leucine aminopeptidases from E. coli, Arabidopsis thaliana, and bovine eye lens revealed that 39.8, 34.9, and 34.0% of the residues were identical, respectively. Residues proposed to be part of the active site or involved in the binding of metal ions in the bovine metalloenzyme were all conserved in R. prowazekii PepA. However, despite the structural and enzymatic similarity to E. coli PepA, the R. prowazekii protein was unable to complement the cer site-specific, PepA-dependent recombination system found in E. coli that resolves ColEl-type plasmid multimers into their monomeric forms.

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“…Resolution of ColEl dimers requires the 280 bp cer site ( Fig. la) and at least four host-encoded proteins: ArgR, PepA, XerC and XerD (Stirling et al, 1988(Stirling et al, , 1989Colloms et al, 1990;Blakely et al, 1993). Consistent with its biological role, cer-Xer recombination is restricted to sites in tandem repeat within the same molecule so that it resolves, but does not generate, multimers.…”

Section: Introductionmentioning

confidence: 99%

Involvement of ArgR and PepA in the pairing of ColE1 dimer resolution sites

Guhathakurta

Summers

1995

Microbiology

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~Dimer formation and associated copy number depression is an important cause of multicopy plasmid instability. Natural multicopy plasmids employ site-specific recombination to convert dimers to monomers, thus maximizing the number of independently segregating molecules at cell division. Resolution of dimers of Escherichia coli plasmid ColEl requires the plasmid cer site and at least four chromosome-encoded proteins: the XerC and XerD recombinases, and accessory factors ArgR and PepA. It has been suggested that ArgR has a role in the initial pairing of recombination sites and we describe here an attempt to detect this process in wivo. Our approach exploits a previous observation that a cefilike site known as the type II hybrid supports inter-molecular recombination and causes extensive multimerization of plasmids. We report that type-ll-mediated multimerization can be repressed by a cer site in cis or in trans and propose that this is due to a physical interaction between the sites. If this hypothesis is correct, suppression of multimer formation provides an assay of site pairing. Our results demonstrate that the putative pairing interaction is independent of the topological relationship of the sites and that both PepA and ArgR are involved. Although most recombination-deficient mutant derivatives of ArgR are unable to pair recombination sites, we have found two (ArgRllO and ArgR115*) which retain pairing activity. The validity of the pairing hypothesis is discussed in the light of alternative explanations for our data.Keywords : cer, plasmid stability, recombination, nucleoprotein complexes, site-specific recombination INTRODUCTIONMulticopy plasmids of Escherichia coli are distributed randomly at cell division and the frequency of plasmid loss increases with decreasing copy number. Plasmid multimer formation reduces the number of independent plasmid molecules in the cell (Summers, 1993;Summers & Sherratt, 1984) and is an important cause of cloning vector instability. Natural, multicopy plasmids such as ColEl avoid this problem because multimers are resolved to monomers by site-specific recombination (Summers & Sherratt, 1984). Resolution of ColEl dimers requires the 280 bp cer site ( Fig. la) and at least four host-encoded proteins: ArgR, PepA, XerC and XerD (Stirling et al., 1988(Stirling et al., , 1989 Colloms et al., 1990;Blakely et al., 1993). Consistent with its biological role, cer-Xer recombination is restricted to sites in tandem repeat within the same molecule so that it resolves, but does not generate, multimers.Of the four proteins required for ColEl multimer resolution, the roles of XerC and XerD are best understood. They are the subunits of a heterodimeric sitespecific recombinase which footprinting experiments have shown to bind to a region of approximately 30 bp between the MlUI and TaqI sites in cer (Blakely e t al., 1993; Fig. la).The roles of ArgR and PepA are less clearly defined but ArgR has been shown to bind specifically to an Arg-box sequence in cer (Stirling e t al., 1988; Fig. 1). PepA, ...

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xerB, an Escherichia coli gene required for plasmid ColE1 site-specific recombination, is identical to pepA, encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase.

Cited by 173 publications

References 33 publications

Isolation and Characterization of the Neutral Leucine Aminopeptidase (LapN) of Tomato

Isolation and Characterization of the Neutral Leucine Aminopeptidase (LapN) of Tomato

Characterization of the Rickettsia prowazekii pepA gene encoding leucine aminopeptidase

Involvement of ArgR and PepA in the pairing of ColE1 dimer resolution sites

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