Direction of Travel of RecBc Recombinase through Bacteriophage Lambda DNA

We examined linkage relationships for RecBC-mediated recombination in lytic cycle crosses of lambda phages bearing two cohesive end sites (cos) oriented in the same direction. The relationships obtained imply that a given recombinant tends to be packaged from the cos site that is the nearer one to the right of the exchange. In view of the previously established coupling of entry of a recombinase at a cos cut and initiation of DNA packaging by that cos cut, these results imply that the recombinase (presumably the RecBC gene product) enters lambda's chromosome at the right end.     

Break-Join Recombination in Phage λ

In phage lambda, when DNA replication is blocked, recombination mediated by the Red pathway occurs only near the double-chain break site, cos, that defines the termini of the virion chromosome. The recombinants initiated by cos contain newly synthesized DNA near cos, in amount corresponding to a few percent of the length of lambda. A restriction enzyme cut delivered to one parent far from cos results in elevated recombination near the restriction site. Recombinants induced by this cut have a similarly small amount of DNA synthesis in these replication-blocked crosses. When restriction cuts are introduced in the presence of normal amounts of all of the DNA replication enzymes, many of the resulting recombinants still enjoy, at most, a small amount of DNA synthesis associated with the exchange event. Thus, these experiments fail to support the previously considered possibility that Red-mediated recombination in lambda proceeds largely through a break-copy pathway.     

Oriented loading of FtsK on KOPS

In Escherichia coli, the ATP-dependent DNA translocase FtsK transports DNA across the site of cell division and activates recombination by the XerCD recombinases at a specific site on the chromosome, dif, to ensure the last stages of chromosome segregation. DNA transport by FtsK is oriented by 8-base-pair asymmetric sequences ('KOPS'). Here we provide evidence that KOPS promote FtsK loading on DNA and that translocation is oriented at this step.     

In phage lambda, cos is a recombinator in the red pathway

Among lambda particles carrying chromosomes that have failed to replicate during a lytic cycle cross there is a high frequency of Red-mediated recombination near the right-hand end. Earlier work has shown that this recombination is dependent on cos (cohesive end site), the packaging origin of lambda. In contrast to the prediction of the break-copy model proposed earlier, we find a high recombination rate near cos even when only one of the two participating parents has a functional cos at that locus. The exchange is accompanied by loss of the stimulating cos in the recombination product, irrespective of the marker configurations: a+b+cos- rather than a+b+cos+ is produced in the cross a+b-cos- x a-b+cos+ as well as in the cross a+b-cos+ x a-b+cos-. Further analyses of these and earlier data allow the formulation of a detailed model for cos-stimulated, Red-mediated genetic exchange. In this model, cos stimulates exchange by virtue of being a double-strand cut site. The model has several features like that proposed for yeast. This role of cos in the Red pathway contrasts with the role of cos in the RecBC pathway, in which cos serves as an entry site for a recombinase that stimulates exchanges far from cos.     

Homologous recombination promoted by Chi sites and RecBC enzyme of Escherichia coli

Chi sites are examples of special sites enhancing homologous recombination in their region of the chromosome. Chi, 5′ G-C-T-G-G-T-G-G3′, is a recognition site for the RecBC enzyme, which nicks DNA near Chi as it unwinds DNA. A molecular model of genetic recombination incorporating these features is reviewed.     

Hotspots for generalized recombination in the Escherichia coli chromosome.

A naturally occurring hotspot for Rec recombination of Escherichia coli was located in the biotin operon. The phenotypes of the bio hotspot as observed in λbio transducing phage were identical to those of Chi mutations in phage λ. In addition to recA⁺ function, the site-specific stimulation of recombination required recB⁺ function. The stimulation took place when the hotspot was present in only one parent of the cross and even when present opposite a region of heterology.The demonstration of a Chi element in E. coli provoked us to measure the density of Chi elements on the chromosome. E. coli DNA sampled in λ transducing phage (either obtained by induction of secondary site lysogens or made in vitro from EcoRI cleavage fragments) showed one hotspot per 5 to 15 × 10³ bases. The high density and the fact that Chi stimulation of recombination can span the inter-Chi distance suggest that Chi might be important in Rec recombination in the absence of λ.     

Does Chi Give or Take?

In lytic cycle crosses with Red-Gam-lambda phage, particles were examined that had undergone an Int-mediated exchange. It was assumed that this exchange dimerized the circular lambda, making it packageable. Among these Int-mediated recombinants, particles were identified that had, in addition, enjoyed a close double exchange mediated by the RecBC pathway. Such close double exchanges indicate localized negative interference and are analogous to eukaryotic conversions that have retained parental configuration of flanking markers. These events are stimulated by Chi, a recombinator specific to the RecBC pathway. When Chi is present in only one parent in the cross, the complementary double exchange recombinants are Chi stimulated to the same degree. This behavior of Chi contrasts with that of characterized eukaryotic recombinators.     

Identification of the Chi site of Haemophilus influenzae as several sequences related to the Escherichia coli Chi site

The Escherichia coli Chi site 5'-GCTGGTGG-3' modulates the activity of the powerful dsDNA exonuclease and helicase RecBCD. Genome sequence analyses revealed that Chi is frequent on the chromosome and oriented with respect to replication on the E . coli genome. Chi is also present much more frequently than predicted statistically for a random 8-mer sequence. Although it is assumed that Chi is ubiquitous, there is virtually no proof that its features are conserved in other microorganisms. We therefore identified and analysed the Chi sequence of an organism for which the full genome sequence was available, Haemophilus influenzae . The biological test we used is based on our finding that rolling circle plasmids provide a specific substrate for RecBCD analogues in different microorganisms. Unexpectedly, several related sequences, corresponding to 5'-GNTGGTGG-3' and 5'-G(G/C)TGGAGG-3', showed Chi activity. As in E . coli , the H . influenzae Chi sites are frequent on the genome, which is in keeping with the need for frequent Chi sites for dsDNA break repair of chromosomal DNA. Although statistically over-represented, this feature is less marked than that of the E . coli Chi site. In contrast to E . coli , the H . influenzae Chi motifs are only slightly oriented with respect to the replication strand. Thus, although Chi appears to have a highly conserved biological role in attenuating exonuclease activity, its sequence characteristics and statistical representation on the genome may differ according to the particular features of the host.     

Processive action of terminase during sequential packaging of bacteriophage lambda chromosomes

Bacteriophage lambda chromosomes are packaged in a polarized, sequential fashion from a multimeric DNA substrate. Mature chromosomes are generated when terminase introduces staggered nicks in the cohesive end sites (cos sites) bounding a chromosome. Packaging is polarized, to the initial and terminal cos sites for packaging a chromosome can be defined. To initiate packaging, terminase binds to cos at cosB, and subsequently cuts at cosN. To terminate packaging of a chromosome, a functional cosB is not required at the terminal cos. To explain this finding, it was proposed earlier that terminase scans for the terminal cosN, rather than any subsequent cosB, during packaging. In the work described here we performed helper packaging experiments to see whether processive action of terminase occurs during sequential packaging of lambda chromosomes. The helper packaging experiments involve trilysogens; strains carrying three prophages in tandem. Infection by a hetero-immune helper phage results in packaging of the repressed prophage chromosomes, since the prophage structure is analogous to the normal DNA substrate. Two chromosomes can be packaged from between the three cos sites of the prophages of a trilysogen. Both chromosomes are packaged even when the central cos is cosB-. Our interpretation of these data is that terminase is brought to the central cos by packaging; following cleavage of the central cos, the terminase remains bound to the distal chromosome; and terminase acts to begin packaging of the distal chromosome. The frequency at which terminase reads across the central cos to initiate packaging of the distal chromosome is in the range from 0.3 to 0.5 in our experiments. Reading across cos was found not to be greatly dependent on the state of cosB, indicating that cosB binding is only needed for packaging the first chromosome in a packaging series. A multilysogen was constructed in which the initial cos was cos+ and the distal cos sites were all cosB-. The initial and downstream chromosomes were found to be packaged. This result indicates that terminase that is brought to the central cos by packaging is not only able to initiate packaging of a downstream chromosome, but can also scan and terminate packaging of the downstream chromosome. A model is presented in which processive action of terminase is the basis for sequential packaging of lambda chromosomes.     

Heteroduplex Chain Polarity in Recombination of Phage λ by the Red, Recbcd, Recbc(d(-)) and Recf Pathways

We have examined the chain polarity of heteroduplex DNA in unreplicated, bacteriophage lambda splice recombinants when recombination was by the RecBCD, RecBC(D-), or RecF pathway of Escherichia coli or the Red pathway of lambda. For each of these pathways, recombination is activated by the cutting of cos that accompanies chromosome packaging, and is effected by recombination enzymes acting at the right end created by that cutting. For exchanges occurring near cos, one parent makes a lesser physical and genetic contribution than does the other. For each pathway, when the phage carried standard cos, this minority contribution was predominantly on the r chain, ending 5' at the right end of lambda. When standard cos was replaced by a cloned inverted cos located centrally on the standard lambda genetic map, minority contribution was predominantly on the l chain. In each case, the polarity of the overlap was usually that formed by 3' overhangs of parental information and material. These results are discussed in the context of current models of recombination for the different pathways.     

Chi sites in combination with RecA protein increase the survival of linear DNA in Escherichia coli by inactivating exoV activity of RecBCD nuclease.

In Escherichia coli, unprotected linear DNA is degraded by exoV activity of the RecBCD nuclease, a protein that plays a central role in the repair of double-strand breaks. Specific short asymmetric sequences, called chi sites, are hotspots for RecBCD-promoted recombination and are shown in vitro to attenuate exoV activity. To study RecBCD-chi site interactions in vivo we used phage lambda's terminase to introduce a site-specific double-strand break at lambda's cos site inserted into a plasmid. We show that after terminase has cut cos in vivo, nucleases degrade linearized DNA only from the end that does not have a strong terminase binding site. Linearized cosmid DNA containing chi sites in the proper orientation to the unprotected end is degraded more slowly in rec+ E. coli than is chi-less DNA. Increased survival of chi-containing DNA is a result of partial inactivation of exoV activity and is dependent on RecA and SSB proteins. The linearization of chi-containing DNA molecules leads to RecA-dependent formation of branched structures which have been proposed as intermediates in the RecBCD pathway of double-strand break repair.     

Chi-dependent intramolecular recombination in Escherichia coli.

Homologous recombination in Escherichia coli is enhanced by a cis-acting octamer sequence named Chi (5''-GCTGGTGG-3'') that interacts with RecBCD. To gain insight into the mechanism of Chi-enhanced recombination, we recruited an experimental system that permits physical monitoring of intramolecular recombination by linear substrates released by in vivo restriction from infecting chimera phage. Recombination of the released substrates depended on recA, recBCD and cis-acting Chi octamers. Recombination proficiency was lowered by a xonA mutation and by mutations that inactivated the RuvABC and RecG resolution enzymes. Activity of Chi sites was influenced by their locations and by the number of Chi octamers at each site. A single Chi site stimulated recombination, but a combination of Chi sites on the two homologs was synergistic. These data suggest a role for Chi at both ends of the linear substrate. Chi was lost in all recombinational exchanges stimulated by a single Chi site. Exchanges in substrates with Chi sites on both homologs occurred in the interval between the sites as well as in the flanking interval. These observations suggest that the generation of circular products by intramolecular recombination involves Chi-dependent processing of one end by RecBCD and pairing of the processed end with its duplex homolog.     

Prophage lambda induces terminal recombination in Escherichia coli by inhibiting chromosome dimer resolution. An orientation-dependent cis-effect lending support to bipolarization of the terminus

A prophage lambda inserted by homologous recombination near dif, the chromosome dimer resolution site of Escherichia coli, is excised at a frequency that depends on its orientation with respect to dif. In wild-type cells, terminal hyper- (TH) recombination is prophage specific and undetectable by a test involving deletion of chromosomal segments between repeats identical to those used for prophage insertion. TH recombination is, however, detected in both excision and deletion assays when Deltadif, xerC, or ftsK mutations inhibit dimer resolution: lack of specialized resolution apparently results in recombinogenic lesions near dif. We also observed that the presence near dif of the prophage, in the orientation causing TH recombination, inhibits dif resolution activity. By its recombinogenic effect, this inhibition explains the enhanced prophage excision in wild-type cells. The primary effect of the prophage is probably an alteration of the dimer resolution regional control, which requires that dif is flanked by suitably oriented (polarized) stretches of DNA. Our model postulates that the prophage inserted near dif in the deleterious orientation disturbs chromosome polarization on the side of the site where it is integrated, because lambda DNA, like the chromosome, is polarized by sequence elements. Candidate sequences are oligomers that display skewed distributions on each oriC-dif chromosome arm and on lambda DNA.     

染色体易位重组位点的自动识别方法研究

染色体易位重组位点的识别对很多染色体遗传性疾病的诊断有着重要的意义.本文基于实际诊断中采集到的24类染色体数据和9号正常与异常染色体数据,构建了一套自动识别染色体易位重组位点的模型和方法.首先,对染色体图像进行预处理,得到了方向梯度直方图特征(HOG)和局部二值模式特征(LBP),构建了基于纹理特征的染色体24分类多通...     

A third defective lambdoid prophage of Escherichia coli K12 defined by the lambda derivative, lambdaqin111

We describe the isolation and characterization of a new Q-independent substitution mutant of lambda, lambdaqin111, which differs from other characterized Q-independent lambda phages. This mutant defines a new lambda-like prophage in the bacterial chromosome, as seen by homologous recombination between lambdaqin111 and the host DNA and by DNA/DNA hybridization methods. Genetic and electron microscopy data show that this new prophage carries, at least, genes analogous to Q-S-R of lambda and also a cos site functionally identical to lambda cos. It is located near 34 min on the Escherichia coli K12 map, i.e. in the same region but at a different site from the defective Rac prophage.     

Chain Bias in Chi-Stimulated Heteroduplex Patches in the λ Ren Gene Is Determined by the Orientation of λ Cos

Heteroduplex patch recombinants have received information in one DNA chain but have not recombined flanking markers. Evidence regarding which chain is exchanged bears on the structure of recombination intermediates. The direction of travel along DNA of RecBCD recombinase, the central enzyme in the Escherichia coli RecBCD pathway of homologous recombination, is determined in phage lambda by the orientation of the packaging origin, cos. cos is a double-chain cut site which serves as a preferred entry site for RecBCD. Using partially denaturing gels to resolve heteroduplex molecules, we have examined patch recombinants at the lambda ren gene. We report that the transferred information in Chi-stimulated patches at ren can occur on either chain, but is biased to the chain ending 5' at the right of the lambda map (the lambda r chain) in phage carrying cos in its normal orientation. The chain bias switches in favor of the chain that ends 3' at the right (the lambda l chain) when RecBCD travel direction is reversed by inverting cos. We entertain models that accommodate these and other results pertaining to the structure of RecBCD-mediated recombinants.     

KOPS-guided DNA translocation by FtsK safeguards Escherichia coli chromosome segregation

The septum-located DNA translocase, FtsK, acts to co-ordinate the late steps of Escherichia coli chromosome segregation with cell division. The FtsK γ regulatory subdomain interacts with 8 bp KOPS DNA sequences, which are oriented from the replication origin to the terminus region ( ter ) in each arm of the chromosome. This interaction directs FtsK translocation towards ter where the final chromosome unlinking by decatenation and chromosome dimer resolution occurs. Chromosome dimer resolution requires FtsK translocation along DNA and its interaction with the XerCD recombinase bound to the recombination site, dif , located within ter . The frequency of chromosome dimer formation is ∼15% per generation in wild-type cells. Here we characterize FtsK alleles that no longer recognize KOPS, yet are proficient for translocation and chromosome dimer resolution. Non-directed FtsK translocation leads to a small reduction in fitness in otherwise normal cell populations, as a consequence of ∼70% of chromosome dimers being resolved to monomers. More serious consequences arise when chromosome dimer formation is increased, or their resolution efficiency is impaired because of defects in chromosome organization and processing. For example, when Cre– loxP recombination replaces XerCD– dif recombination in dimer resolution, when functional MukBEF is absent, or when replication terminates away from ter .     

On the birth and fate of bacterial division sites

Thanks to genetics, to the study of protein–protein interactions and to direct viewing of subcellular structures by the use of immunofluorescence and green fluorescent protein (GFP) fusions, the organization of the constriction apparatus of walled bacteria is gradually coming to light. The tubulin-like protein FtsZ assembles as a ring around the site of constriction and operates as an organizer and activator of septum-shaping proteins. Much less is known about the factors specifying the location of FtsZ rings. Circumstantial evidence favours the presence at future ring positions of fixed elements, the potential division sites (PDS), before FtsZ assembles. FtsZ polymerization is initiated from a point on a PDS, the nucleation site, still to be identified, and proceeds bidirectionally around the cell. We hypothesize that new PDS are specified in a manner that depends on the functioning of an active chromosome partition apparatus. This view is supported by the fact that formation of mid-cell PDS requires initiation of DNA replication, and by recent studies supporting the existence of a specialized partition apparatus in a variety of microorganisms. Although PDS may be specified directly by the partition apparatus, indirect localization linked to compartmentalized gene expression during chromosome segregation is also possible. Once created, PDS are used in a regulated manner, and several mechanisms normally operate to direct constriction to selected PDS at the correct time. One, dedicated to the permanent suppression of polar PDS, rests on the minicell suppression system and involves a protein that is able to discriminate between polar and non-polar sites. Another is involved in asymmetric site selection at the early stages of sporulation in Bacillus subtilis . Finally, a mechanism observed only in certain multinucleated cells appears to favour division at non-polar PDS related to the most ancient replication/DNA segregation events.     

The relaxed requirements of the integron cleavage site allow predictable changes in integron target specificity

Integrons are able to incorporate exogenous genes embedded in mobile cassettes, by a site-specific recombination mechanism. Gene cassettes are collected at the attI site, via an integrase mediated recombination between the cassette recombination site, attC, and the attI site. Interestingly, only three nucleotides are conserved between attC and attI. Here, we have determined the requirements of these in recombination, using the recombination machinery from the paradigmatic class 1 integron. We found that, strikingly, the only requirement is to have identical first nucleotide in the two partner sites, but not the nature of this nucleotide. Furthermore, we showed that the reaction is close to wild-type efficiency when one of the nucleotides in the second or third position is mutated in either the attC or the attI1 site, while identical mutations can have drastic effects when both sites are mutated, resulting in a dramatic decrease of recombination frequency compared to that of the wild-type sites. Finally, we tested the functional role of the amino acids predicted from structural data to interact with the cleavage site. We found that, if the recombination site triplets are tolerant to mutation, the amino acids interacting with them are extremely constrained.