Single-stranded regions in Streptococcus pneumoniae chromosomal deoxyribonucleic acid and their relation to transformation.

Deoxyribonucleic acid (DNA) in lysates of both completent and noncompetent streptococcus pneumoniae cells was characterized by chromatography on benzoylated, naphthoylated diethylaminoethyl-cellulose columns, by sensitivity to Aspergillus oryzae S1 endonuclease, and by sucrose gradient analysis. The DNAs from both competent and noncompetent cells were found to contain similar extents of single-stranded regions. These single-stranded regions appeared to be intact, unpaired regions in double-stranded DNA rather than gaps, nicks, or unpaired ends in the DNA. Inhibition of cells with rifampin prior to lysis increased the amount of such single strandedness in the DNA. Lysates made at various times after [14C]thymidine-labeled cells had bound [3H]thymidine-labeled transforming DNA were also characterized by benzoylated, naphthoylated diethylaminoethyl-cellulose chromatography. Changes in the elution profiles of DNA from cells exposed to homospecific (S. pneumoniae) donor DNA were indicative of the formation of complexes between donor DNA and the single-stranded regions of recipient DNA. In contrast, profiles of DNA from cells exposed to heterospecific (S. sanguis) DNA did not show significant changes, indicating that few such donor-recipient complexes were formed during heterospecific transformation.     

Effect of ethylenediaminetetraacetic acid on deoxyribonucleic acid entry and recombination in transformation of a wild-type strain and a rec-1 mutant of Haemophilus influenzae

In transformation of Haemophilus influenzae, donor deoxyribonucleic acid (DNA) enters into competent cells in the presence of ethylenediaminetetraacetic acid (EDTA), which prevents the formation of single stranded regions in the donor DNA that has entered. If after entry of DNA the recipient cells were first incubated at 17 degrees C and then at 37 degrees C in the continuous presence of EDTA, almost no integration occurred. On the other hand, if after entry of DNA the cells were incubated first at 17 degrees C in the absence of EDTA, allowing the generation of single-stranded regions (integration is blocked at this temperature), and then at 37 degrees C in the presence of EDTA, donor-recipient DNA complexes were formed. These results suggest that single-stranded regions are required for integration. Integration to completion was strongly inhibited by EDTA. In a rec-1 mutant of H. influenzae no donor-recipient DNA complexes carrying recombinant-type activity were formed during incubation at 37 degrees C in the absence of EDTA. If rec-1 cells were incubated at 37 degrees C in the presence of EDTA, which strongly inhibited breakdown of DNA, donor-recipient DNA complexes were formed if previously single-stranded regions in the donor DNA that had entered were generated by incubation at 17 degrees C in the absence of EDTA. This suggests that the rec-1 protein protects the initial donor-recipient DNA complex against degradation, so that further steps in the recombination process can proceed.     

Fate of heterospecific transforming DNA bound to Streptococcus sanguis.

The fate of 3H-labeled str-r fus-s DNA from Streptococcus pneumoniae, bound after a 1-min uptake to 14C-labeled str-s fus-r S. sanguis recipients, was followed by techniques previously developed for analyzing the fate of homospecific DNA. Heterospecific S. pneumoniae DNA was bound and formed complexes with recipient protein in a manner similar to that of homospecific DNA but transformed relatively poorly. The rate at which complexed heterospecific DNA becomes physically associated with recipient DNA, and at which donor markers are integrated into the chromosome, was slower than in the case of homospecific DNA. In addition, about half of the heterospecific donor counts initially bound in trichloracetic acid-insoluble form were gradually solubilized and released from the cell. The association of heterospecific DNA with the recipient chromosome was more unstable than that involving homospecific DNA, since only associations of the former type were largely dissociated by isolation and resedimentation. The donor DNA-containing material so dissociated had the same sedimentation properties as complexed heterospecific DNA before association, indicating that the complex of single-stranded donor DNA and recipient protein formed on uptake moves as a whole from its site of formation to synapse with the chromosome.     

Mechanism of transformation in B. subtilis

Summary Transformation in B. subtilis is achieved by the uptake of donor DNA into recipient cells and the integration of part of this donor DNA into the host chromosome. The evidence presented in this report is interpreted to indicate that donor double helical DNA, on entry into host cells is rapidly membrane bound and can remain in this state for a consicerable time, perhaps even until integration. This bound DNA consists of molecules which have been reduced in size and degraded on uptake, and appear as partially single-stranded molecules. It is suggested that the donor DNA initially forms single strands which rapidly assume a partially single stranded nature by association with the host DNA or by reannealing.Host cells, by virtue of the competent state, possess temporarily, and prior to the addition of donor DNA, chromosomes with single-stranded gaps. It is likely that such gaps are larger than the single-stranded segments of donor DNA which are to be integrated. Results are described which are best explained if integration is achieved by an initial annealing between the single-stranded donor and host segments followed by their covalent linkage.     

Fate of homospecific transforming DNA bound to Streptococcus sanguis.

The fate of [3H]DNA from Streptococcus sanguis str-r43 fus-s donors in [14C]S. sanguis str-s fus-r1 recipients was studied by examining the lysates prepared from such recipients at various times after 1 min of exposure to DNA. The lysates were analyzed in CsCl and 10 to 30% sucrose gradients; fractions from the gradients were tested for biological activity and sensitivity to nucleases, subjected to various treatments and retested for nuclease sensitivity, and run on 5 to 20% neutral and alkaline sucrose gradients. The results demonstrate that donor DNA bound to S. sanguis cells in a form resistant to exogenous deoxyribonuclease is initially single stranded and complexed to recipient material. Donor DNA can be removed from the complex upon treatment of the complex with Pronase, phenol, or isoamyl alcohol-chloroform. Within the complex, donor DNA is relatively insensitive to S1 endonuclease but can regain its sensitivity by treatment with phenol. With time the complex moves as a whole to associate physically with the recipient chromosome. After a noncovalent stage of synapsis, donor material is covalently bonded to and acquires the nuclease sensitivity of recipient DNA, while donor markers regain transforming activity and become linked to resident markers.     

The presence of single-stranded regions in mammalian DNA

Benzoylated naphthoylated DEAE-cellulose chromatography has been used in this study to demonstrate the presence of single-stranded regions in the DNA of mouse L cells and Chinese hamster ovary cells. In mouse L cell DNA the majority of the single-stranded regions were found to have free ends, as judged from their susceptibility to S₁ endonuclease and exonuclease VII. They were separated on average by a distance of 70 μm.The single-stranded regions fell into two classes, distinguished by whether or not they were derived from the replication fork. Pulse-label administered to Chinese hamster ovary cells was found to be closely associated with the single-stranded regions, as shearing the DNA left a greater proportion of the pulse-label than the bulk-label in the single-strand-containing fraction. However, reasonable estimates of the number of replication forks present at one time showed that the single-stranded regions occurred too frequently along the DNA for them all to be associated with replication. When DNA was isolated from Chinese hamster ovary cells synchronized in G₁ phase, it too was found to contain single-stranded regions, separated by a distance of 100 μm. The regions in the G₁ cells were demonstrated not to be the result of a low level of DNA replication nor to be an artifact of the isolation procedure. Possible functions of this non-replicative class of single-stranded regions are discussed. They may, for example, act as regulatory signals or to be pre-formed initiation points for DNA replication. They are the subject of ongoing investigation.     

Transformation in Bacillus subtilis: biological and physical evidence for a novel DNA-intermediate in synchronously transforming cells.

Summary Competent B. subtilis cells exposed to transforming DNA in the presence of EDTA bind, but do not take up DNA. Rapid and almost synchronous uptake of the bound DNA is achieved by the addition of Mg²⁺ ions in excess of the EDTA. At 30° and at 17° comparable numbers of transformants are produced from cells pre-locaded with DNA at 30° (after termination of uptake by the addition of DNA ase the samples were incubated at 37°). However, almost no transformants are produced when cells are exposed to DNA at 17°, although binding does take place then. Because DNA is taken up at 17° after having loaded the cells at 30°, whereas no uptake occurs after binding at 17°, it is suggested that binding of DNA to the cellular surface involves at least two steps.In DNA re-extracted from cells at 17°, pre-loaded with DNA at 30°, little recombinant type activity is present, indicating that integration is blocked at 17°. However, physico-chemical analysis of the reextracted DNA indicates that a complex between single-stranded donor DNA and the recipient chromosome is formed at 17°. This complex has a higher buoyant density than donor-recipient complexes formed at 30°.     

Single-stranded conjugation in Escherichia coli K-12

Summary The mutation BT43 in the gene dnaB leads to the inhibition of vegetative and conjugational DNA synthesis at 42°. The consequences in case of conjugation are very unusual. The fragment of donor DNA tramsmitted to the recipient cell remains single-stranded and is integrated as such into the recipient chromosome similar to the main events during transformation. We call this process single-stranded (SS) conjugation.The evidence for this statement comes from the measurement of the time of expression of the gene tsx, containing the genetic information for the receptor of phage T6. The gene tsx is introduced into a dnaBT43 recipient cell alternatively by two different donors Hfr H and Hfr C, which are characterized by opposite directions of transfer. Therefore both donors introduce into the recipient cell alternatively the informational or noninformational DNA strand. If conjugation is performed at a nonpermissive temperature, the transferred DNA piece remains single-stranded and is integrated as such into the recipient chromosome. If it is the informational strand (case of Hfr H), it is transcribed very fast and yields the protein in question in about 20 min. If the noninformational strand is integrated (Hfr C) about 40 min additional time is required to effect cell division.SS-conjugation is very sensitive to the action of exonucleases Exo I and Exo V and is much enhanced in the absence of both nucleases in the recipient.The exogenous DNA pieces are integrated as short insertions, this leads to the disjoining of linked markers and to a very short scale of the genetic map. Because the donor DNA undergoes recombination in the single-stranded state heteroduplex regions originate which are subsequently corrected by the enzymes of the recipient cell. The situation leads to a very special but predictable heterogeneity of the progeny of transconjugants.The fact of the existence of this special process, SS-conjugation, drastically different from common conjugation in many respects, suggests that common conjugation leads to the integration of double-stranded DNA pieces into the recipient chromosome.     

Mechanism of conjugation and recombination in bacteria. XVII. Further evidence for single-stranded regions in recipient DNA during conjugation in Escherichia coli K-12.

The secondary structure of recipient DNA mated with Hfr strain was investigated by CsCl density gradient fractionation. After 45 min of HfrH64 X 3h-f-ab1157 mating one-fourth of the radioactive recipient DNA was recovered as a single-strand but only after shearing of cell lysates prior to centrifugation. This heavier than native DNA fraction of radioactive material (obtained after the first centrifugation) was degraded by single-strand specific nuclease S from Aspergillus oryzae. These findings thus confirm the authors' earlier results suggesting that in the course of mating are generated local single-stranded regions in recipient DNA.     

An unstable donor-recipient DNA complex in transformation of Bacillus subtilis.

Summary In re-extracted DNA obtained shortly after uptake of transforming DNA by Bacillus subtilis, increased amounts of donor DNA radioactivity banding at the position of donor-recipient DNA complex (DRC) are observed in CsCl gradients, if the cells are irradiated with high doses of UV prior to reextraction of the DNA. Qualitatively, the same phenomenon is observed if lysates of transforming cells are irradiated. UV-irradiation of lysates of competent cells to which single-stranded DNA is added after lysis, does not result in linkage of this DNA to the chromosomal DNA. Two observations argue in favour of the formation of a specific labile complex between donor and resident DNA during transformation. Firstly, heterologous donor DNA from Escherichia coli, although being processed to single-stranded DNA in competent B. subtilis, does not seem to be linked to the recipient chromosome upon UV-irradiation, and secondly, the labile complex of donor and recipient DNA can be stabilized by means of treatment of the lysates of transforming cells with 4, 5¹, 8-trimethylpsoralen in conjuction with long-wave ultra violet light irradiation. This indicates that base-pairing is involved in the formation of the complex. On the basis of these results we assume that the unstable complex of donor and recipient DNA is an early intermediate in genetic recombination during transformation.     

Characterization of transposon-generated protease mutant of xanthomonas campestris pathovar glycine 8ra

Protease negative mutant of Xanthomonas campestris pathovar glycine 8ra (prt-mutant) was constructed by mutagenesis employing artificial transposon Omegon-Km. Transposon delivery was conducted through diparental conjugation using X. campestris pathovar glycine 8ra as recipient and Escherichia coli S17-1 carrying pJFF 3500 plasmid as the donor. The frequency of transconjugation was found 1.9 x 10(-7) per recipient. Enzyme analysis indicated the presence of mutant with lower protease activity than that of the wild-type. Genetic analysis employing pulsed-field gel electrophoresis (PFGE) of the genomic DNA digested with AseI or SpeI restriction endonuclease could significantly differentiate X. campestris pathovar glycine 8ra prt from the wild-type parent. The 9.85 kb pLR omega 6 plasmid was constructed from the genomic DNA of the prt mutant after being digested with KpnI restriction endonuclease and ligated with T4 DNA ligase.     

Specific hydrolysis of the cohesive ends of bacteriophage lambda DNA by three single strand-specific nucleases.

Procedures have been worked out for Aspergillus nuclease S1 and mung been nuclease to quantitatively cleave off both of the 12-nucleotide long, single-stranded cohesive ends of lambdaDNA. This cleavage is indicated by the almost complete elimination of the repair incorporation of radioactive nucleotides by DNA polymerase into the digested DNA. With S1 nuclease, cleavage was complete at 10 degrees as well as at 30 degrees. Under the conditions for quantitative cleavage of the single-stranded regions there was no digestion of the double-stranded lambdaDNA. The mung bean nuclease cleaved off the cohesive ends completely at 30 degrees but at 5 degrees, the cleavage was not complete even at high enzyme concentration. The nearest neighbor analysis of the repaired DNA indicates that at 5 degrees about four nucleotides remained undigested. The mung bean nuclease also introduced, under the conditions used, some nicks into double-stranded DNA as determined by the repair incorporation. The Escherichia coli exonuclease VII cleaved off part of the cohesive ends of lambdaDNA, leaving two nucleotides on each end as single-stranded tails.     

Synthesis of murine leukemia viral DNA in vitro: evidence for plus-strand DNA synthesis at both ends of the genome

We studied the synthesis of B-tropic murine leukemia viral DNA in vitro by detergent-disrupted virions. The reaction products (detected by the Southern transfer technique) included full-length, infectious, double-stranded DNA and several subgenomic fragments. Restriction endonuclease analysis and hybridization and specific probes revealed two classes of subgenomic fragments: some were derived from the right end of the genome, and some were derived from the left end. Most of the fragments harbored one long terminal repeat copy at their ends, suggesting that they were initiated correctly. S1 nuclease and restriction endonuclease treatments of these fragments indicated that a single-stranded gap was present near the first initiation site of plus strong-stop DNA. The treatments also suggested the presence of a second initiation site flanked by a single-stranded gap 0.9 kilobase pairs from the right end of the genome. Our data clearly show that plus-strand DNA is synthesized at both ends of the genome, by using plus strong stop as the first initiation site and additional initiation sites.     

The Saccharomyces cerevisiae Msh2 mismatch repair protein localizes to recombination intermediates in vivo

Mismatch repair proteins act during double-strand break repair (DSBR) to correct mismatches in heteroduplex DNA, to suppress recombination between divergent sequences, and to promote removal of nonhomologous DNA at DSB ends. We investigated yeast Msh2p association with recombination intermediates in vivo using chromatin immunoprecipitation. During DSBR involving nonhomologous ends, Msh2p localized strongly to recipient and donor sequences. Localization required Msh3p and was greatly reduced in rad50delta strains. Minimal localization of Msh2p was observed during fully homologous repair, but this was increased in rad52delta strains. These findings argue that Msh2p-Msh3p associates with intermediates early in DSBR to participate in the rejection of homeologous pairing and to stabilize nonhomologous tails for cleavage by Rad1p-Rad10p endonuclease.     

Transformation in Bacillus subtilis: characterization of an intermediate in recombination observed during transformation.

Summary From recombination-proficient competent cells of Bacillus subtilis in which the donor DNA entered at 17°, and which were kept at the same temperature, a complex of donor DNA and the recipient chromosome can be obtained which has a relatively high buoyant density in CsCl gradients. Exposure of the isolated complex to nuclease S1 liberates donor radioactivity. The limited biological activity of DNA re-extracted from cells attempting to recombine at 17° is decreased upon incubation with nuclease S1. If recombination is allowed to proceed at 30°, the high buoyant density of the donor-recipient complex decreases to normal values and less radioactivity can be liberated from the complex by nuclease S1. Concomitantly the biological activity of re-extracted DNA becomes less vulnerable to nuclease S1 under these conditions. On the basis of these observations we assume that the intermediate complex partly consists of unpaired single-stranded donor DNA.Support for the correctness of this assumption is derived from experiments with a mutant, which is delayed in the processing of high buoyant density donor-recipient complex to normal buoyant density donor-recipient complex. This delay is reflected in the time of acquisition of resistance to nuclease S1 digestion of the isolated complex.     

Intracellular effects of phage phi W-14 DNA on transformation of Bacillus subtilis

Summary Uptake of transforming DNA by competent Bacillus subtilis cells in the presence of phage W-14 DNA (in which half the thymine residues are replaced by -putrescinyl-thymine) is accompanied by a decrease in the amount of trichloracetic acid-precipitable label of the former retained by recipient cells during subsequent incubation. Fractionation of lysates of cells incubated for 0.5 min at 37°C after DNA uptake at 30°C in the presence of low concentrations of W-14 DNA (0.1 g/ml) demonstrated the presence of single-stranded transforming DNA molecules, typical for DNA taken up by B. subtilis. The intracellular effect of W-14 DNA was enhanced by an increase in its concentration (to 0.5–1 g/ml), or by increasing the temperature of uptake (to 37°C). With either of these treatments transforming DNA taken up was found in the form of a broad asymmetric band, indicative of degradation, and partially located at the density characteristic for single-stranded molecules. Fractionation of lysates of cells treated (0.1 g/ml) or untreated with W-14 DNA, and incubated for 20 min at 37°C after DNA uptake, showed disappearance of the single-stranded band. Donor DNA label was then found exclusively in the recipient DNA band, its amount being lower in samples treated with W-14 DNA. The influence of a high concentration of W-14 DNA on retention of transforming DNA label was correlated with its effect on transformation. On exposure to low concentrations of phage DNA, such a correlation was observed only after longer periods of incubation, due to slower intracellular degradation of homologous DNA taken up. The results are consistent with the proposal that W-14 DNA-induced reduction in efficiency of transformation is due to intracellular stimulation of transforming DNA degradation, leading to a decrease in the number of donor molecules available for recombination with the recipient chromosome.     

Molecular fate of heterologous bacterial DNA in competent Bacillus subtilis: further characterization of unstable association between donor and recipient DNA and the involvement of the cellular membrane

Summary Although heterospecific transformation is extremely inefficient and very little heterologous donor DNA integrates into the recipient chromosome in a stable way, we have previously shown that B. pumilus DNA entering competent B. subtilis efficiently associates with the recipient chromosome in an unstable way. This association can be stabilized by photocrosslinking in the presence of 4,5,8-trimethylpsoralen; it depends on the recombination proficiency of the recipient strain and on strand-separation of the recipient chromosome (te Riele and Venema 1982b). The present study provides further evidence that the heterologous donor DNA and the recipient DNA are associated by regions of base-pairing. Based on the high sensitivity of the donor moiety in the complex to nuclease S1 (90%) and the high sensitivity of the complex to moderate denaturing conditions (Tm=48°C), we presume that donor and recipient DNA are associated either by several short sequences of 15–25 fairly well matched base pairs or by a region of base-pairing of about 200 bases, which contains 25% of mismatches. During incubation, the unstable complex disappears, probably due to nucleolytic degradation.The unstable heterologous donor-recipient complex (DRC) was found to be membrane-bound. However, in contrast to homologous DRC, the unstable heterologous DRC remains membrane bound during incubation. Apparently, the predominantly single-stranded character of the heterologous DRC prevents release of the complex from the membrane.Abbreviations DRC donor-recipient complex - TMP 4,5,8-trimethyl-psoralen - DNAase I deoxyribonuclease 1 - TCA trichloroacetic acid     

Molecular Fate of Heterologous Bacterial DNA in Competent BACILLUS SUBTILIS. II. Unstable Association of Heterologous DNA with the Recipient Chromosome

In CsCl density gradients of lysates from competent Bacillus subtilis cells, which had been exposed to heterologous bacterial DNA, very little donor-recipient complex (DRC) formation could be detected. The present study demonstrates that photocrosslinking of such lysates by irradiation with long-wave UV light in the presence of 4,5',8-trimethylpsoralen results in a dramatic increase in the amount of heterologous DRC. This phenomenon may be interpreted as the stabilization of a pre-existing weak association between entered heterologous donor DNA and one recipient strand in unpaired regions of the chromosome. When a recombination-deficient mutant is used, the amount of stabilizable heterologous DRC is reduced to the same extent as the specific transforming activity of homologous DNA. Although the amount of stabilizable complex is related to the degree of homology between donor and recipient DNA, this relation is not a quantitative one. Probably the association is caused by very short regions of base pairing between the donor and recipient moieties in the complex. Heating of a lysate at 70° prior to photocrosslinking prevents stabilization, apparently because the regions of base pairing are rapidly melted out. The results described in this paper can be best interpreted as the fixation of a process in which entered donor DNA in competent cells tries to find homologous stretches in the recipient chromosome.     

Fate of heterologous deoxyribonucleic acid in Bacillus subtilis.

CsCl density gradient fractionation of cell lysates was employed to follow the fate of Escherichia coli, phage T6, and non-glucosylated phage T6 deoxyribonucleic acid (DNA) after uptake by competent cells of Bacillus subtilis 168 thy minus trp minus. Shortly after uptake, most of the radioactive Escherichia coli or non-glucosylated T6 DNA was found in the denatured form; the remainder of the label was associated with recipient DNA. Incubation of the cells after DNA uptake led to the disappearance of denatured donor DNA and to an increase in the amount of donor label associated with recipient DNA. These findings are analogous to those previously reported with homologous DNA. By contrast, T6 DNA, which is poorly taken up, appeared in the native form shortly after uptake and was degraded on subsequent incubation. The nature of the heterologous DNA fragments associated with recipient DNA was investigated with Escherichia coli 2-H and 3-H-labeled DNA. Association of radioactivity with recipient DNA decreased to one-fourth in the presence of excess thymidine; residual radioactivity could not be separated from recipient DNA by shearing (sonic oscillation) and/or denaturation, but was reduced by one-half in the presence of a DNA replication inhibitor. Residual radioactivity associated with donor DNA under these conditions was about 5% of that originally taken up. Excess thymidine, but not the DNA replication inhibitor, also decreased association of homologous DNA label with recipient DNA; but, even in the presence of both of these, the decrease amounted to only 60%. It is concluded that most, or all, of the Escherichia coli DNA label taken up is associated with recipient DNA in the form of mononucleotides via DNA replication.