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PubMed1997

Direct interaction of aminopeptidase A with recombination site DNA in Xer site-specific recombination.

Xer site-specific recombination at ColE1 cer converts plasmid multimers into monomers, thus ensuring the heritable stability of ColE1. Two related recombinase proteins, XerC and XerD, catalyse the strand exchange reaction at a 30 bp recombination core site. In addition, two accessory proteins, PepA and ArgR, are required for recombination at cer. These two accessory proteins are thought to act at 180 bp of accessory sequences adjacent to the cer recombination core to ensure that recombination only occurs between directly repeated sites on the same molecule. Here, we demonstrate that PepA and ArgR interact directly with cer, forming a complex in which the accessory sequences of two cer sites are interwrapped approximately three times in a right-handed fashion. We present a model for this synaptic complex, and propose that strand exchange can only occur after the formation of this complex.

Amino Acid Sequence↗

PubMed1988

Genetic control of intrachromosomal recombination in Saccharomyces cerevisiae. I. Isolation and genetic characterization of hyper-recombination mutations.

Eight complementation groups have been defined for recessive mutations conferring an increased mitotic intrachromosomal recombination phenotype (hpr genes) in Saccharomyces cerevisiae. Some of the mutations preferentially increase intrachromosomal gene conversion (hpr4, hpr5 and hpr8) between repeated sequences, some increase loss of a marker between duplicated genes (hpr1 and hpr6), and some increase both types of events (hpr2, hpr3 and hpr7). New alleles of the CDC2 and CDC17 genes were recovered among these mutants. The mutants were also characterized for sensitivity to DNA damaging agents and for mutator activity. Among the more interesting mutants are hpr5, which shows a biased gene conversion in a leu2-112::URA3::leu2-k duplication; and hpr1, which has a much weaker effect on interchromosomal mitotic recombination than on intrachromosomal mitotic recombination. These analyses suggest that gene conversion and reciprocal exchange can be separated mutationally. Further studies are required to show whether different recombination pathways or different outcomes of the same recombination pathway are controlled by the genes identified in this study.

Cell Cycle↗

PubMed1993

Efficient double-strand break-stimulated recombination promoted by the general recombination systems of phages lambda and P22.

To examine bacteriophage recombination in vivo, independent of such other processes as replication and packaging, substituted lambda phages bearing restriction site polymorphisms were employed in a direct physical assay. Bacteria were infected with two phage variants; DNA was extracted from the infected cells and cut with a restriction endonuclease. The production of a unique recombinant fragment was measured by Southern blotting and hybridization with a substitution sequence-specific probe. High frequency recombination was observed under the following conditions: the substituted lambda phages infected a wild-type host cell bearing a lambda repressor-expressing plasmid designed to shut down phage transcription and inhibit phage DNA replication as well. The same plasmid expressed the lambda red and gam genes. In addition, the host cell bore a second plasmid which expressed the EcoRI restriction-modification system. Both phage chromosomes possessed a single EcoRI site in the middle of the marked substitution sequence; however, as the site was modified in one of the parent phages, only the other partner was cut. Recombination was found to be dependent upon (1) red, (2) recA, (3) inactivation of the host recBCD function, either by Gam protein or by mutation and (4) double-strand breaks. The homologous recombination system of phage P22 could substitute for that of lambda.

Bacteriophage P22↗

PubMed1995

Marker-dependent recombination in T4 bacteriophage. IV. Recombinational effects of antimutator T4 DNA polymerase.

Recombinational effects of the antimutator allele tsL42 of gene 43 of phage T4, encoding DNA polymerase, were studied in crosses between rIIB mutants. Recombination under tsL42-restricted conditions differed from the normal one in several respects: (1) basic recombination was enhanced, especially within very short distances; (2) mismatch repair tracts were shortened, while the contribution of mismatch repair to recombination was not changed; (3) marker interference at very short distances was augmented. We infer that the T4 DNA polymerase is directly involved in mismatch repair, performing both excision of a nonmatched single strand (by its 3'-->5' exonuclease) and filling the resulting gap. A pathway for the mismatch repair was substantiated; it includes sequential action of endo VII (gp49)-->3'-->5' exonuclease (gp43)-->DNA polymerase (gp43)-->DNA ligase (gp30). It is argued that the marker interference at very short distances may result from the same sequence of events during the final processing of recombinational intermediates.

Alleles↗

PubMed1996

Meiotic recombination initiated by a double-strand break in rad50 delta yeast cells otherwise unable to initiate meiotic recombination.

Meiotic recombination in Saccharomyces cerevisiae is initiated by double- strand breaks (DSBs). We have developed a system to compare the properties of meiotic DSBs with those created by the site-specific HO endonuclease. HO endonuclease was expressed under the control of the meiotic-specific SPO13 promoter, creating a DSB at a single site on one of yeast's 16 chromosomes. In Rad+ strains the times of appearance of the HO-induced DSBs and of subsequent recombinants are coincident with those induced by normal meiotic DSBs. Physical monitoring of DNA showed that SPO13: : HO induced gene conversions both in Rad+ and in rad50 delta cells that cannot initiate normal meiotic DSBs. We find that the RAD50 gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad50 delta cells, some DSBs are not repaired until a broken chromosome has been packaged into a spore and is subsequently germinated. This suggests that a broken chromosome does not signal an arrest of progression through meiosis. The recombination defect in rad50 delta diploids is not, however, meiotic specific, as mitotic rad50 diploids, experiencing an HO-induced DSB, exhibit similar departures from wild-type recombination.

DNA, Fungal↗

PubMed1997

Unraveling a region-specific hyper-recombination phenomenon: genetic control and modalities of terminal recombination in Escherichia coli.

The propensity of the terminus of the Escherichia coli chromosome for recombination has been further explored, using a test based on the selectable loss of a lambda prophage inserted between repeated sequences from Tn10. Terminal recombination appears region-specific and unrelated to replication termination in a strain harboring a major chromosomal rearrangement. It requires RecBC(D) activity and must therefore occur between sister chromosomes, to conserve genomic integrity in spite of DNA degradation by RecBCD. Terminal recombination is maximal in the dif region and its intensity on either side of this recombination site depends on the orientation of the repeated sequences, probably because of the single chi site present in each repeat. Additional observations support the model that the crossover is initiated by single-strand invasion between sister chromosomes followed by RecBCD action as a consequence of DNA breakage due to the initial invasion event. Crossover location within repeats inserted at dif position supports the possibility that sister chromosomes are tightly paired in the centre of the terminal recombination zone. These data reinforce the model that postreplicative reconstruction of nucleoid organization creates a localized synapsis between the termini of sister chromosomes.

Chromosomes, Bacterial↗

PubMed2003

On the determination of recombination rates in intermated recombinant inbred populations.

The recurrent intermating of F(2) individuals for some number of generations followed by several generations of inbreeding produces an intermated recombinant inbred (IRI) population. Such populations are currently being developed in the plant-breeding community because linkage associations present in an F(2) population are broken down and a population of fixed inbred lines is also created. The increased levels of recombination enable higher-resolution mapping in IRI populations relative to F(2) populations. Herein we derive relationships, under several limiting assumptions, for determining the expected recombination fraction in IRI populations from the crossover rate per meiosis. These relationships are applicable to situations where the inbreeding component of IRI population development is by either self-fertilization or full-sib mating. Additionally, we show that the derived equations can be solved for the crossover rate per meiosis if the recombination fraction is known for the IRI population. Thus, the observed recombination fraction in any IRI population can be expressed on an F(2) basis. The implications of this work on the expansion of genetic maps in IRI populations and limits for detecting linkage between markers are also considered.

Chromosome Mapping↗

PubMed1997

The role of recombination signal sequences in the preferential joining by deletion in DH-JH recombination and in the ordered rearrangement of the IgH locus.

The bias favoring deletion over inversion in DH-JH rearrangement has been known for years, but the underlying mechanism has yet to be fully defined. It has been suggested that the ratio of deletion/inversion is determined by the combined effect of two factors: (i) the relative strengths of 5' and 3' recombination signal sequences (RSS) of a DH segment, and (ii) the efficiency with which the deletional product (one joint) forms relative to the inversional product (two joints). In this study, we analyzed for the first time the effect of factor 1 alone on the biased 3' RSS utilization in DH-JH joining by using deletional plasmids in an extrachromosomal substrate V(D)J recombination assay. It was found that the 3' RSS and associated coding end (12 bp) mediate recombination more efficiently than the 5' RSS/coding end DH-JH plasmids. These results demonstrate that the effect of the RSS/coding end alone can account, at least partially, for the predominant deletion in DH-JH recombination. The potential effect of the relative strength of RSS and associated coding end on the ordered rearrangement of DH-JH followed by VH to DH-JH was also assessed. When recombination frequencies of D-->J (3' DH to J3) were compared with frequencies of V-->D (VHPJ14 to 3' DH or VHOX2 to 3' DH), it was found that V-->D joining was, if anything, more efficient than D-->J joining. Therefore, if all three segments were accessible, RSS/coding end effects would not contribute to the ordered rearrangement of the IgH locus.

Abelson murine leukemia virus↗

PubMed1993

Two cDNAs from the plant Arabidopsis thaliana that partially restore recombination proficiency and DNA-damage resistance to E. coli mutants lacking recombination-intermediate-resolution activities.

Escherichia coli ruvC recG mutants lack RuvC endonuclease, which resolves crossed-strand joint molecules (Holliday junctions) formed during homologous recombination into recombinant products, and an activity (RecG) thought to partially replace RuvC. They are therefore highly deficient in homologous recombination, and sensitive to UV light and chemical DNA-damaging agents, presumably because of inability to tolerate unrepaired DNA damage by recombinational mechanisms (Lloyd, R.G. (1991) J. Bacteriol. 173:5414-5418). We transformed these mutants with plasmids expressing cDNAs from the plant Arabidopsis thaliana. Selection for bacteria with increased resistance to methylmethanesulfonate yielded two cDNAs, designated DRT111 and DRT112 (DNA-damage-repair/toleration). Expression of these plant cDNAs, especially DRT111, restored conjugal recombination proficiencies in ruvC and ruvC recG mutants to nearly wild-type levels. Both plant cDNAs significantly increased resistance of both mutants to UV light and several chemical DNA-damaging agents, but did not fully correct the mutant phenotypes. Drt111 activity, but not Drt112, also increased, to nearly wild-type levels, resistance of recG single mutants to UV plus mitomycin C. The predicted Drt111 and Drt112 polypeptides, 383 and 167 amino acids respectively, show no similarity with one another or with prokaryotic Holliday resolvases. Both appear chloroplast targeted; Drt112 is highly homologous to Arabidopsis plastocyanin. DRT111 and DRT112 probes hybridize only to DNA from closely related plants.

Arabidopsis↗

PubMed1996

Site-specific recombination by the beta protein from the streptococcal plasmid pSM19035: minimal recombination sequences and crossing over site.

The beta recombinase from the broad host range Grampositive plasmid pSM19035 catalyzes intramolecular site-specific recombination between two directly or inversely oriented recombination sites in the presence of a chromatin-associated protein (Hbsu). The recombination site had been localized to a 447 bp DNA segment from pSM19035. This segment includes a 90 bp region that contains two adjacent binding sites (I and II) for beta protein dimers. Using in vitro recombination assays, we show that this 90 bp region is necessary and sufficient for beta protein-mediated recombination; this defines the six site as the region required for beta protein binding. The point of crossing over has been localized to the center of site I. Hbsu has a strong binding affinity for an unknown site located within the 447 bp segment containing the six site. We discuss the possibility that Hbsu recognizes an altered DNA structure, rather than a specific sequence, generated in the synaptic complex.

Bacterial Proteins↗

PubMed2003

High prevalence of diverse forms of HIV-1 intersubtype recombinants in Central Myanmar: geographical hot spot of extensive recombination.

OBJECTIVES: To investigate the molecular epidemiology and genetic structure of HIV-1s causing the epidemic in Central Myanmar and to explore the genesis of HIV epidemic in this area. DESIGN: A molecular epidemiological investigation was conducted in 1999-2000 in the city of Mandalay among high-risk populations and the structural features of circulating HIV-1s were analyzed. METHODS: HIV-1 genotypes of 59 specimens were screened based on gag (p17) and env (C2/V3) regions. Near full-length nucleotide sequences of HIV-1 isolates with subtype discordance were determined and their recombinant structures were characterized. RESULTS: Three lineages of HIV-1 strains, including CRF01_AE (27, 45.8%), subtype B' (Thailand variant of subtype B) (15, 25.4%) and subtype C (8, 13.6%), were distributed in Mandalay, while substantial portions (9, 15.3%) of specimens showed various patterns of subtype discordance in different regions of HIV-1 genomes. The study on six HIV-1 isolates with subtype discordance revealed that they were highly diverse types of unique recombinant forms (URFs) comprised of various combinations of three circulating subtypes. One URF was a particularly complex mosaic that contained 13 recombination breakpoints between three HIV-1 subtypes. Approximately half of recombinants showed 'pseudotype' virion structures, in which the external portions of envelope glycoproteins were exchanged with different lineages of HIV-1 strains, suggesting the potential selective advantage of 'pseudotype' viruses over parental strains. CONCLUSION: The study revealed the unique geographical hot spot in Central Myanmar where extensive recombination events appeared to be taking place continually. This reflects the presence of highly exposed individuals and social networks of HIV-1 transmission.

Adolescent↗

PubMed1995

The role of chromatin-associated protein Hbsu in beta-mediated DNA recombination is to facilitate the joining of distant recombination sites.

The beta recombinase is unable to mediate in vitro DNA recombination between two directly oriented recombination sites unless a bacterial chromatin-associated protein (Bacillus subtilis Hbsu or Escherichia [correction of Eschrichia] coli HU] is provided. By electron microscopy, we show that the role of Hbsu is to help in joining the recombination sites to form a stable synaptic complex. Some evidence supports the fact that Hbsu works by recognizing and stabilizing a DNA structure at the recombination site, rather than by serving as a bridge between beta recombinase dimers through a protein-protein interaction. We show that the mammalian HMG1 protein, which shares neither sequence nor structural homology with Hbsu, can also stimulate beta-mediated recombination. These chromatin-associated proteins share the property of binding to DNA in a relatively non-specific fashion, bending it, and having a marked preference for altered DNA structures. Hbsu, HU or HMG1 proteins probably bind specifically at the crossing-over region, since at limiting protein-DNA molar ratios they could not be outcompeted by an excess of a DNA lacking the crossing over site. Distamycin, a minor groove binder that induces local distortions in DNA, did not affect the binding of beta protein to DNA, but inhibited the formation of the synaptic complex.

Bacterial Proteins↗

PubMed1979

B-cell alloantigens in two recombinant families: prediction of a B/D recombination using B-cell-specific alloantisera.

During the VIIth Histocompatibility Workshop, 44 sera were selected that defined B-cell alloantigens showing correlation with the HLA--D antigenic determinants. The segregation of these antigens, now called DRw (HLA--D-related), was studied in a family that contained one HLA--A/B recombinant and one HLA--B/D recombinant sibling. It could be established that the DRw-1 antigenic determinants segregated with the B-D region in the A/B recombinant sibling and that the DRw-2 antigen segregated with the D region of the B/D recombinant child. In the second family, DRw typing segregated with the D region of the B/D recombinant child. In the second family, DRw typing showed that one of the children was identical with an HLA--ABC non-identical sibling. This suggested that a crossing-over had occurred. This was confirmed by mutual non-stimulation of these HLA--ABC non-identical children in the mixed lymphocyte culture. This crossing-over involved the DRw-3 alloantigen. These data confirm the assumption that serologically defined B-cell alloantigens are coded for by genes located outside the HLA--A/B region on the B-D side of chromosome. Moreover, these data suggest that, within families, serological B-cell genotype identity can predict a mutual unresponsiveness in the lymphocyte culture. However, findings in these two families indicate that discrepancies nevertheless exist between B-cell serology and mixed lymphocyte reactions and that mutual stimulation can occur despite apparent identity of serological B-cell phenotype and that B-cell phenotype identity does not necessarily predict mutual unresponsiveness in the mixed lymphocyte culture.

Adolescent↗

PubMed1990

Molecular analysis of two recombinant mouse strains with crossovers in the E alpha recombination hot spot.

Recombination in the E alpha gene of the mouse major histocompatibility complex has been found to occur only in mice derived from crosses between strains with the k and p haplotypes. In the present paper the crossover sites of six additional strains were examined by RFLP analysis. These recombinant strains were derived from crosses between strains (B10.RPD1, B10.RPD2) with crossovers in the E alpha gene and B10.M (H-2f). Four have crossover sites between the E alpha gene and the S region. One recombinant (B10.RFD7) also crossed over within the E alpha gene and a second (B10.RPF1) crossed over within a segment located to the left of the E alpha gene. This study suggests that the DNA sequence responsible for recombination in the E alpha gene is also present in the B10.RPD1 and B10.RPD2 recombinant strains.

Animals↗

PubMed1984

Recombinant cold-adapted attenuated influenza A vaccines for use in children: reactogenicity and antigenic activity of cold-adapted recombinants and analysis of isolates from the vaccinees.

Reactogenicity and antigenic activity of recombinants obtained by crossing cold-adapted donor of attenuation A/Leningrad/134/47/57 with wild-type influenza virus strains A/Leningrad/322/79(H1N1) and A/Bangkok/1/79(H3N2) were studied. The recombinants were areactogenic when administered as an intranasal spray to children aged 3 to 15, including those who lacked or had only low titers of pre-existing anti-hemagglutinin and anti-neuraminidase antibody in their blood. After two administrations of vaccines at a 3-week interval, both strains induced antibody in 75 to 95% of the children. On coinfection of chicken embryos with both recombinants, only weak interference was observed. Administration to children of the bivalent vaccine containing H1N1 and H3N2 recombinants induced efficient production of antibody to H1 and H3 hemagglutinins and N1 and N2 neuraminidases without adverse reactions. The recombinants studied were genetically stable as judged by retention of the temperature-sensitive phenotypes and a lack of reversion of the genes carrying temperature-sensitive mutations in all of the reisolates from vaccinated children.

Adolescent↗

PubMed1978

Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes.

Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny. All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold. Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107). Some differences in transduction proficiency were also observed. Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain. The results indicate that genetic recombination in E. coli is a highly regulated process involving multiple gene products.

DNA Repair↗

PubMed1989

Site-specific recombination between cloned attP and attB sites from the Haemophilus influenzae bacteriophage HP1 propagated in recombination-deficient Escherichia coli.

Plasmids were constructed which contain both attP and attB DNA segments derived from the insertion sites of the lysogenic bacteriophage HP1 and its host, Haemophilus influenzae. Similar plasmids containing the two junction segments (attL and attR regions) between the phage genome and the lysogenic host chromosome were also prepared. The formation of recombinant dimer plasmids was observed when attP-attB plasmids were propagated in Escherichia coli HB101 (recA), while plasmids containing the junction segments did not form recombinant dimers. Deletion of the phage DNA segment adjacent to the attP site from the attP-attB constructions eliminated detectable recombination, suggesting that this sequence contains the gene encoding the HP1 integrase. No plasmid recombination was observed in strains of E. coli defective in integration host factor. This suggests that integration host factor is important in the expression or activity of the system which produces the site-specific recombination of sequences derived from HP1 and H. influenzae. Further, it suggests that a protein functionally analogous to E. coli integration host factor may be present in H. influenzae.

Bacteriophages↗

PubMed1997

Role of enzymes of homologous recombination in illegitimate plasmid recombination in Bacillus subtilis.

The structural stability of plasmid pGP1, which encodes a fusion between the penicillinase gene (penP) of Bacillus licheniformis and the Escherichia coli lacZ gene, was investigated in Bacillus subtilis strains expressing mutated subunits of the ATP-dependent nuclease, AddAB, and strains lacking the major recombination enzyme, RecA. Strains carrying a mutation in the ATP-binding site of the AddB subunit exhibited high levels of plasmid instability, whereas a comparable mutation in the A subunit did not affect plasmid stability. Using an alternative plasmid system, pGP100, we were able to demonstrate that the differences in stability reflected differences in initial recombination frequencies. Based on a comparison of endpoint sequences observed in the various hosts, we speculate that at least two different mechanisms underlie the deletion events involved, the first (type I) occurring between nonrepeated sequences, and the second (type II) occurring between short direct repeats (DRs). The latter event was independent of single-strand replication intermediates and the mode of replication and possibly requires the introduction of double-strand breaks (DSBs) between the repeats. In the absence of functional AddAB complex, or the AddB subunit, DSBs are likely to be processed via a recA-independent mechanism, resulting in intramolecular recombination between the DRs. In wild-type cells, such DSBs are supposed to be either repaired by a mechanism involving AddAB-dependent recombination or degraded by the AddAB-associated exonuclease activity. Plasmid stability assays in a recA mutant showed that (i) the level of deletion formation was considerably higher in this host and (ii) that deletions between short DRs occurred at higher frequencies than those described previously for the parental strain. We propose that in wild-type cells, the recA gene product is involved in recombinational repair of DSBs.

Adenosine Triphosphate↗