Isolation, by tetracycline selection, of small plasmids derived from R-factor R12 in Escherichia coli K-12.

The examination, by agarose gel electrophoresis, of tetracycline-resistant colonies of Escherichia coli K-12 carrying R-factor R12 reveals the presence of smaller plasmid deoxyribonucleic acids (DNAs), incompatible with R12, in many of the clones. These plasmids are demonstrated to be homologous with R12 DNA by electron microscope heteroduplex experiments and by the production of consistent fragment patterns upon digestion with various restriction endonucleases. These autonomously replicating plasmids form a related series of covalently closed circular DNA molecules ranging in size from 3.6 X 10(6) to 61 X 10(6) daltons. Plasmids of molecular weight between 3.6 X 10(6) and 37 X 10(6) confer no antibiotic resistances, but when jointly present with R12 by nonetheless enhance the expression of the tetracycline resistance associated with this latter molecule.     

Dimers of escherichia coli F' factors

Covalently closed circular DNA dimers of several $\text{E.}$$\text{coli}$ sex factors have been isolated. One of these, F′451, a dimer of F′450, has a molecular weight of $\text{ca.}$ 230 × 10⁶ daltons. F′451 (λ) containing a λ prophage has a molecular weight of 260 × 10⁶ and is probably the largest covalent closed circle of DNA yet reported. These dimers arise spontaneously and are of unknown origin and significance.     

Heteroduplex mapping of small plasmids derived from R-factor R12: in vivo recombination occurs at IS1 insertion sequences.

Small, autonomously replicating plasmids derived by in vivo recombination from R-factor R12 (= R100) have been structurally mapped by heteroduplex formation between the plasmids and an R-factor which is structurally closely related to R6-5. Recombination resulting in generation of the small resistance-free plasmids occurs between the (IS1)b insertion sequence and various other sites on the opposite side of an origin of replication. A larger R12-derived plasmid pSM17, carrying streptomycin (Sm), sulfadiazole (Sa), and chloramphenicol (Cm) resistances, has recombined in a similar manner but at the (IS1)a sequence. A new structural coordinate origin for R100 and for partially homologous R-factors is proposed based upon the location of the (IS1)b sequence.     

Transduction of R Factors by a Proteus mirabilis Bacteriophage

A transducing phage, designated phim, was isolated from a lysogenic strain of Proteus mirabilis and was characterized with respect to its physical and genetic properties. The phage contains double-stranded deoxyribonucleic acid (DNA) with an S(20,w) degrees of 29 which corresponds to a molecular weight of 24 x 10(6) daltons. The base composition of phim DNA was estimated to be 40% guanine plus cytosine on the basis of the buoyant density of the DNA. phim carries out generalized transduction of chromosomal genes in P. mirabilis at a frequency of 5 x 10(-8) to 2 x 10(-6) per adsorbed phage. To obtain R-factor transduction, it was necessary to have a resident R factor in the recipient cells. In these experiments, different combinations of genetically distinguishable R factors were used in the donor and recipient cells. The frequencies of R-factor transduction were 10(-9) to 2 x 10(-8). The transduction of R factors using an R(-) recipient could not be detected. Transductant R factors were usually recombinant between donor and resident R factors. All of the transduced R factors were transferable by conjugation. A plausible explanation for the requirement for a resident R factor in the recipient cells is that phim transduces only a portion of the R-factor genome and therefore requires a resident R factor for genetic recombination. The reason for the low frequencies of R-factor transduction is not known, but some possible interpretations have been discussed.     

Molecular organization of plasmid R906 (Inc P-1)

Genetic and restriction (for enzymes EcoRI, BamHI and HindIII) maps of the relatively broad host range plasmid R906 are constructed. There are two non-essential regions on the R906 DNA which can be deleted and cloned. Non-essential regions confer a resistance to different agents and restriction sites are clustered in these regions. Essential and conjugativity genes are located in two other DNA regions approximately at 0-23 and 29-44 kb of the R906 map. These large regions share a high level of homology with Inc-1 group plasmids R751 and RP4 according to Southern-blot hybridization and heteroduplex analyses. A transposon-like structure is found on the R751 DNA among R751/R906 heteroduplex molecules. This transposon of total length 5.1 kb has 1.4 kb inverted repeats at the ends. Bla genes of R906 and RP4 plasmids do not have homologous sequences. Data evidence that IncP-1 group plasmids irrespective to their original bacterial source and range of coded antibiotic resistance have very similar molecular organization. The role of possible factors which are responsible for the broad host range property of the IncP-1 group plasmids is discussed.     

Molecular nature of two beta-lactamase-specifying plasmids isolated from Haemophilus influenzae type b.

The molecular nature of two beta-lactamase-specifying plasmids isolated from two separate ampicillin-resistant Haemophilus influenzae type b strains was examined. A 30 X 10(6)-dalton (30-Mdal) plasmid (RSF007) had a copy number of approximately 3 per chromosomal equivalent and a mole fraction guanine plus cytosine content of 0.39. By heteroduplex analysis the 30-Mdal plasmid was found to contain the entire ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. A 3.0-Mdal plasmid (RSF0885) was found as a multicopy pool of approximately 28 copies per chromosomal equivalent, had a mole fraction guanine plus cytosine content of 0.40, and contained only about one-third of the transposable TnA sequence. RSF007 and RSF0885 appeared to be unrelated plasmids in that they share base sequence homology only within the confines of the TnA segment. The 3.0-Mdal Haemophilus plasmid was used to transform E. coli to ampicillin resistance but was found to be unstable in this host in the absence of antibiotic. The possibility that R-plasmids arose in Haemophilus by the translocation of TnA from a donor R-factor onto an indigenous H. influenzae plasmid is discussed.     

Loss and repair of conjugal fertility and infectivity of the resistance factor and sex factor in Escherichia coli

Hirota, Yukinori (University of Osaka, Osaka, Japan), Toshio Fujii, and Yukinobu Nishimura. Loss and repair of conjugal fertility and infectivity of the resistance factor and sex factor in Escherichia coli. J. Bacteriol. 91:1298-1304. 1966.-The drug-resistance factor, R, and the sex factor, F, have homologous traits, including contagious transmission, mediation of sexuality of the host cell, and autonomous replication in their host bacteria. Cooperation between F and R factors was found with a mutant R factor, which is nontransmissible in F(-) bacteria, becoming transmissible when introduced into bacteria carrying F. Conversely, the chromosome of a sterile male strain carrying the mutant sex factor, F(r), becomes transmissible when an R factor is introduced into the cell. The genetic determinants of R factors have been analyzed by isolation of mutant R factors, by sexual conjugation of the host bacteria, and by transduction of R factors with phage P1kc. The fertility determinant of the R factor, m, is inseparable from the determinant for its infectivity, but can be separated from the loci for autonomous replication of the R factor. R and F thus carry genetic determinants governing the same functions.     

RAD54 controls access to the invading 3′-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae

Rad51 is a key protein in homologous recombination performing homology search and DNA strand invasion. After DNA strand exchange Rad51 protein is stuck on the double-stranded heteroduplex DNA product of DNA strand invasion. This is a problem, because DNA polymerase requires access to the invading 3′-OH end to initiate DNA synthesis. Here we show that, the Saccharomyces cerevisiae dsDNA motor protein Rad54 solves this problem by dissociating yeast Rad51 protein bound to the heteroduplex DNA after DNA strand invasion. The reaction required species-specific interaction between both proteins and the ATPase activity of Rad54 protein. This mechanism rationalizes the in vivo requirement of Rad54 protein for the turnover of Rad51 foci and explains the observed dependence of the transition from homologous pairing to DNA synthesis on Rad54 protein in vegetative and meiotic yeast cells.     

Homology between the deoxyribonucleic acid of fertility factor P and Vibrio cholerae chromosomal deoxyribonucleic acid.

The deoxyribonucleic acid (DNA) of the Vibrio cholerae fertility factor P was isolated by the dye-buoyant density method and hybridized to V. cholerae chromosomal DNA. The DNA of this fertility plasmid had between 35 to 40% homology with the V. cholerae chromosomal DNA. Little or no homology was detected between the P factor DNA and DNA of the Escherichia coli sex factor F.     

Induction of recombination between homologous and diverged DNAs by double-strand gaps and breaks and role of mismatch repair.

Sequence homology is expected to influence recombination. To further understand mechanisms of recombination and the impact of reduced homology, we examined recombination during transformation between plasmid-borne DNA flanking a double-strand break (DSB) or gap and its chromosomal homolog. Previous reports have concentrated on spontaneous recombination or initiation by undefined lesions. Sequence divergence of approximately 16% reduced transformation frequencies by at least 10-fold. Gene conversion patterns associated with double-strand gap repair of episomal plasmids or with plasmid integration were analyzed by restriction endonuclease mapping and DNA sequencing. For episomal plasmids carrying homeologous DNA, at least one input end was always preserved beyond 10 bp, whereas for plasmids carrying homologous DNA, both input ends were converted beyond 80 bp in 60% of the transformants. The system allowed the recovery of transformants carrying mixtures of recombinant molecules that might arise if heteroduplex DNA--a presumed recombination intermediate--escapes mismatch repair. Gene conversion involving homologous DNAs frequently involved DNA mismatch repair, directed to a broken strand. A mutation in the PMS1 mismatch repair gene significantly increased the fraction of transformants carrying a mixture of plasmids for homologous DNAs, indicating that PMS1 can participate in DSB-initiated recombination. Since nearly all transformants involving homeologous DNAs carried a single recombinant plasmid in both Pms+ and Pms- strains, stable heteroduplex DNA appears less likely than for homologous DNAs. Regardless of homology, gene conversion does not appear to occur by nucleolytic expansion of a DSB to a gap prior to recombination. The results with homeologous DNAs are consistent with a recombinational repair model that we propose does not require the formation of stable heteroduplex DNA but instead involves other homology-dependent interactions that allow recombination-dependent DNA synthesis.     

Radioprobing of a RecA-three-stranded DNA complex with iodine 125: evidence for recognition of homology in the major groove of the target duplex

A fundamental problem in homologous recombination is how homology between DNAs is recognized. In all current models, a recombination protein loads onto a single strand of DNA and scans another duplex for homology. When homology is found, a synaptic complex is formed, leading to strand exchange and a heteroduplex. A novel technique based on strand cleavage by the Auger radiodecay of iodine 125, allows us to determine the distances between (125)I on the incoming strand and the target sugars of the duplex DNA strands in an Escherichia coli RecA protein-mediated synaptic complex. Analysis of these distances shows that the complex represents a post-strand exchange intermediate in which the heteroduplex is located in the center, while the outgoing strand forms a relatively wide helix intertwined with the heteroduplex and located in its minor groove. The structure implies that homology is recognized in the major groove of the duplex.     

The ami locus of the Gram-positive bacterium Streptococcus pneumoniae is similar to binding protein-dependent transport operons of Gram-negative bacteria

The complete nucleotide sequence of the ami locus of Streptococcus pneumoniae revealed the presence of six open reading frames, amiABCDEF. The predicted Ami proteins are probably involved in a transport system. The AmiA, C, D, E, and F proteins exhibit homology with components of the oligopeptide permeases (opp) of Salmonella typhimurium and Escherichia coli. Intriguingly, the AmiB protein is homologous to ArsC, a cytosolic modifier subunit of the anion pump encoded by the arsenical resistance operon of the R-factor R773 from E. coli. Data are presented which indicate that Ami is indeed a transport system.     

Evolution of a Site of Specific Genetic Homology on the Chromosome of Escherichia coli

Integration of the factors F(v) and F into the chromosome of a substrain of Escherichia coli K-12 has been studied. The F(v) factor is a fertility factor derived from Col V, lacking the ability to govern the production of colicin V. The derivatives of an Hfr(v) (Hfr isolated from a V colicinogenic parent) strain, PK2 (initially isolated from C600 V(+)), were shown to retain a unique bidirectional sex factor affinity locus between recA and pheA. This site shows no affinity for the E. coli K-12 F factor as shown by inability to isolate Hfr strains with origins in this region from a parental strain containing a cytoplasmic F factor. However this area exhibits two regions of homology to the V colicinogenic factor. One gives rise to Hfr(v) strains identical to the original Hfr(v) strain, PK2, with an origin and polarity of transfer designated pheA-CC injecting markers in the order pheA-his-trp-pro. The second gives rise to strains apparently originating at the same site but with reverse polarity designated recA-C, transferring markers in the order recA-thyA-str-xyl. For strains possessing the F(v) factor only the second homology is apparent. A model for the evolution of these strains is presented.     

Molecular studies of FIme resistance plasmids, particularly in epidemic Salmonella typhimurium.

Summary The molecular sizes of F₁ me resistance plasmids from strains of Salmonella typhimurium, S. wien and S. typhi were within the range 87.9–102.6×10⁶ daltons. DNA reassociation studies indicated that the plasmids from these hosts had at least 80% of their nucleotide sequences in common. A high proportion of F₁ me plasmids cannot mediate their own transfer. The non-autotransferring property of such plasmids is the result of DNA deletion; a non-autotransferring F₁ me plasmid was about 10×10⁶ daltons shorter than autotransferring representatives of the group, and its DNA showed 100% homology with them. Plasmids of the F₁ me group are incompatible with the F factor and with F₁R factors. F₁ me plasmids are incompatible with the fi ⁺ MP10 plasmid of S. typhimurium, whereas F and F₁ factors are compatible with MP10 (Anderson et al., 1977). There was no significant DNA homology between members of the F₁ me group and MP10, and these plasmids may share only a small region of DNA responsible for their incompatibility. The F₁ me R factors examined had 29–37% DNA homology with the F factor, and 50–58% homology with the F₁ resistance plasmid, R162. Molecular examination therefore supports the genetic differentiation of members of the F₁ me group from other F-like plasmids. Both types of investigation can thus be used in epidemiological studies of bacterial strains carrying resistance or other plasmids.     

R-factor trimethoprim resistance mechanism: an insusceptible target site

R-factor R388 increases the resistance of $\text{Escherichia coli}$ to trimethoprim by 10,000 fold, and mediates the synthesis of an addional dihydrofolate reductase that is less susceptible to trimethoprim by a similar order of magnitude. The dihydrofolate reductase conferred by the R-factor was of a larger molecular weight than the wild-type enzyme and exhibited a different pattern of response to trimethoprim inhibition. This is thought to be the first example of an R-factor conferring an altered target site mechanism of resistance to a chemotherapeutic agent.     

Comparisons of F Factors and R Factors: Existence of Independent Regulation Groups in F Factors

The similarity of sex pili mediated by F factors and R(fi(+)) factors and the ability of R(fi(+)) factors to control by repression the functioning of pilus genes encoded by the F factor suggested that F factors and R(fi(+)) factors are closely related. Further comparisons of the episomal properties of F factors and R(fi(+)) factors, however, indicated many differences. F factors contain information for a restriction system for phages phiII and T7. Cells containing R factors are sensitive to these phages. Furthermore, R(fi(+)) factors do not repress the F factor phiII restriction system in cells containing both an R(fi(+)) factor and an F factor. R factors and F factors are heteroimmune episomes. In addition, an R(fi(+)) factor in cells containing both an R factor and an F factor does not fully repress the expression of F-factor immunity to an incoming second F factor. R-factor and F-factor replication systems are not identical. Wild-type F-factor replication genes will complement the mutant F(ts114)lac(+) replication genes in cells containing two F factors. The F(ts114)lac(+) episome is retained when these cells are grown at 42 C; however, cells containing an R(fi(+)) factor and F(ts114)lac(+) lose the F(ts114)lac(+) when grown at 42 C, at the same rate as cells containing only the F(ts114)lac(+). The replication system of the R(fi(+)) factor will not complement the mutant F(ts114)lac(+) replication system.     

The poor homology stringency in the heteroduplex allows strand exchange to incorporate desirable mismatches without sacrificing recognition in vivo

RecA family proteins are responsible for homology search and strand exchange. In bacteria, homology search begins after RecA binds an initiating single-stranded DNA (ssDNA) in the primary DNA-binding site, forming the presynaptic filament. Once the filament is formed, it interrogates double-stranded DNA (dsDNA). During the interrogation, bases in the dsDNA attempt to form Watson–Crick bonds with the corresponding bases in the initiating strand. Mismatch dependent instability in the base pairing in the heteroduplex strand exchange product could provide stringent recognition; however, we present experimental and theoretical results suggesting that the heteroduplex stability is insensitive to mismatches. We also present data suggesting that an initial homology test of 8 contiguous bases rejects most interactions containing more than 1/8 mismatches without forming a detectable 20 bp product. We propose that, in vivo, the sparsity of accidental sequence matches allows an initial 8 bp test to rapidly reject almost all non-homologous sequences. We speculate that once the initial test is passed, the mismatch insensitive binding in the heteroduplex allows short mismatched regions to be incorporated in otherwise homologous strand exchange products even though sequences with less homology are eventually rejected.     

A catenated DNA molecule as an intermediate in the replication of the resistance transfer factor R6K in Escherichia coli

Pulse-labeling of an $\text{Escherichia coli}$ strain harboring the resistance transfer factor R6K results in a transient increase in labeled catenated R6K DNA molecules. After a chase the level of labeled catenated DNA molecules is greatly reduced concomitant with a marked increase in labeling of the supercoiled DNA form of R6K. The data presented support a role for the catenated DNA molecule as an intermediate in the replication of the plasmid R6K.     

DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

The formation of heteroduplex DNA is a central step in the exchange of DNA sequences via homologous recombination, and in the accurate repair of broken chromosomes via homology-directed repair pathways. In cells, heteroduplex DNA largely arises through the activities of recombination proteins that promote DNA-pairing and annealing reactions. Classes of proteins involved in pairing and annealing include RecA-family DNA-pairing proteins, single-stranded DNA (ssDNA)-binding proteins, recombination mediator proteins, annealing proteins, and nucleases. This review explores the properties of these pairing and annealing proteins, and highlights their roles in complex recombination processes including the double Holliday junction (DhJ) formation, synthesis-dependent strand annealing, and single-strand annealing pathways—DNA transactions that are critical both for genome stability in individual organisms and for the evolution of species.A central step in the process of homologous recombination is the formation of heteroduplex DNA. In this article, heteroduplex DNA is defined as double-stranded DNA that arose from recombination, in which the two strands are derived from different parental DNA molecules or regions. The two strands of the heteroduplex may be fully complementary in sequence, or may contain small regions of noncomplementarity embedded within their otherwise complementary sequences. In either case, Watson-Crick base pairs must stabilize the heteroduplex to the extent that it can exist as free DNA following the dissociation of the recombination proteins that promoted its formation.The ability to form heteroduplex DNA using strands from two different parental DNA molecules lies at the heart of fundamental biological processes that control genome stability in individual organisms, inheritance of genetic information by their progeny, and genetic diversity within the resulting populations (Amunugama and Fishel 2012). During meiosis, the formation of heteroduplex DNA facilitates crossing-over and allelic exchange between homologous chromosomes; this process ensures that progeny are not identical clones of their parents and that sexual reproduction between individuals will result in a genetically diverse population (see Lam and Keeney 2015; Zickler and Kleckner 2015). Heteroduplex DNA generated by meiotic COs also ensures proper segregation of homologous chromosomes, so that each gamete receives a complete but genetically distinct set of chromosomes (Bascom-Slack et al. 1997; Gerton and Hawley 2005). In mitotic cells, heteroduplex DNA formation between sister chromatids is essential for homology-directed repair (HR) of DNA double-strand breaks (DSBs), stalled replication forks, and other lesions (Maher et al. 2011; Amunugama and Fishel 2012; Mehta and Haber 2014). Prokaryotic organisms also generate heteroduplex DNA to perform HR transactions, and to promote genetic exchanges, such as occur during bacterial conjugation (Cox 1999; Thomas and Nielsen 2005).Fundamentally, heteroduplex DNA generation involves the formation of tracts of Watson-Crick base pairs between strands of DNA derived from two different progenitor (parental) DNA molecules. Mechanistically, the DNA transactions giving rise to heteroduplex may involve two, three, or four strands of DNA (Fig. 1). DNA annealing refers to heteroduplex formation from two complementary (or nearly complementary) molecules or regions of single-stranded DNA (ssDNA) (Fig. 1A). DNA annealing may occur spontaneously, but it is promoted in vivo by certain classes of annealing proteins. Three-stranded reactions yielding heteroduplex DNA proceed by a different mechanism referred to as DNA pairing, strand invasion, or strand exchange. These reactions involve the invasion of a duplex DNA molecule by homologous (or nearly homologous) ssDNA. The invading DNA may be completely single stranded, as is often the case in in vitro assays for DNA-pairing activity (Fig. 1B) (Cox and Lehman 1981). Under physiological conditions, however, the invading ssDNA is contained as a single-stranded tail or gap within a duplex (Fig. 1C,D). DNA-pairing reactions are promoted by DNA-pairing proteins of the RecA family (Bianco et al. 1998), and proceed via the formation of D-loop or joint molecule intermediates that contain the heteroduplex DNA (Fig. 1B–D). Three-stranded reactions may also be promoted by exonuclease/annealing protein complexes found in certain viruses. Four-stranded reactions generating heteroduplex DNA involve branch migration of a Holliday junction (Fig. 1D). In practice, a four-stranded reaction must be initiated by a three-stranded pairing reaction catalyzed by a DNA-pairing protein, after which the heteroduplex is extended into duplex regions through the action of the DNA-pairing protein or of an associated DNA helicase/translocase (Das Gupta et al. 1981; Kim et al. 1992; Tsaneva et al. 1992).Open in a separate windowFigure 1.Common DNA annealing and pairing reactions. (A) Simple annealing between two complementary molecules of single-stranded DNA to form a heteroduplex. (B) Three-stranded DNA-pairing reaction of the type used for in vitro assays of RecA-family DNA-pairing proteins. The single-stranded circle is homologous to the linear duplex. Formation of heteroduplex (red strand base-paired to black) requires protein-promoted invasion of the duplex by the ssDNA to form a joint molecule or D-loop (i). The length of the heteroduplex may be extended by branch migration (ii). (C) Three-stranded DNA-pairing reaction of the type used for high-fidelity repair of DNA DSBs in vivo. The invading strand is the ssDNA tail of a resected DSB. The 3′ end of the invading strand is incorporated into the heteroduplex within the D-loop intermediate. (D) Example of a four-stranded DNA-pairing transaction that is initiated by a three-stranded pairing event and extended by branch migration. The ssDNA in a gapped duplex serves as the invading strand to generate a joint molecule (i), reminiscent of the reaction shown in panel B. Protein-directed branch migration may proceed into the duplex region adjacent to the original gap, generating α-structure intermediates (ii), or eventually a complete exchange of strands (iii).     

R-Factor Mutant Capable of Specifying Hypersynthesis of Penicillinase

The physical characteristics of a mutant, R(M201-2), capable of conferring high and stable ampicillion resistance was analyzed. The R(M201-2) and its parent R-factor deoxyribonucleic acid (DNA) could be isolated as an extrachromosomal and covalently closed circular form. Their buoyant densities were both 1.712 g/cm(3), and their molecular weights were about 82 x 10(6) and 64 x 10(6), respectively, when measured by CsCl and sucrose density gradient analyses. The contour lengths by electron microscopy were 35.9 +/- 0.6 and 31.0 +/- 0.6 mum, respectively. By using the extracted R-factor DNA, the mutant and parent characters were transformable to another Escherichia coli strain. The mutant R factor showed an increased amount of DNA even after conjugal transfer to Proteus. An increase in the size of R-factor DNA was thus considered to be the cause of the high level of ampicillin resistance.