Unstable association in vitro between donor and recipient DNA in Bacillus subtilis

Randen, J van; Wiersma, K; Venema, Gerard

doi:10.1128/jb.152.1.275-283.1982

J Bacteriol

1982

DOI: 10.1128/jb.152.1.275-283.1982

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Unstable association in vitro between donor and recipient DNA in Bacillus subtilis

J van Randen¹,

K Wiersma²,

Gerard Venema³

Abstract: In addition to stable donor-recipient DNA complexes, unstable complexes between donor and recipient DNA were formed in vitro with Bacillus subtilis. Whereas the stable complexes survived CsCl gradient centrifugation at pH 11.2 and phenol plus sodium p-aminosalicylate extraction with 0.17 M NaCl, the unstable complexes dissociated during these manipulations. The donor moiety from the unstable complexes remained associated with the recipient DNA during phenol plus sodium p-aminosalicylate treatment at 0.85 M NaC… Show more

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Cited by 3 publications

References 20 publications

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Initiation of recombination during transformation of Bacillus subtilis requires no extensive homologous sequences

Randen¹,

Wiersma²,

Venema³

1982

Mol Gen Genet

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Lysates obtained shortly after entry of transforming DNA to Bacillus subtilis contain donor-recipient DNA complexes, in which the donor moiety is associated with the recipient DNA in an unstable way. The complexes could be artificially stabilized by crosslinking with 4,5',8-trimethylpsoralen. The unstable complexes dissociated upon helix-destabilizing treatments, such as heating at 70 degrees C, and CsCl gradient centrifugation at pH 11.2, but remained stable during CsCl gradient centrifugation at pH 10. Donor-recipient DNA complexes were not formed after entry of heterologous pUB110 DNA. These observations suggest that base-pairing is involved in the unstable association. The donor moiety of the unstable complexes was completely, or almost completely, digestible by nuclease S1, indicating that the donor and recipient base-sequences are only paired over very short distances. The unstable donor-recipient DNA complexes are true recombination intermediates because (i) strain 7G224 (recE4) was impaired in the formation of the unstable complexes, and (ii) the unstable complexes were rapidly converted to stable complexes in recombination proficient strains, whereas their conversion was delayed in the recombination deficient strain 7G84. Unstable complexes were also formed with Escherichia coli donor DNA, but to a lesser extent. Apparently a limited degree of base-sequence homology is sufficient to initiate recombination.

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Initiation of recombination during transformation of Bacillus subtilis requires no extensive homologous sequences

Randen¹,

Wiersma²,

Venema³

1982

Mol Gen Genet

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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

Riele

Venema

1984

Mol Gen Genet

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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 degrees 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.

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Involvement of single-strand breaks in complex formation between single-stranded DNA and nucleoids of Bacillus subtilis

J¹,

Wiersma²,

Venema³

1984

Molec. Gen. Genet.

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RNase-unfolded chromosomes of competent Bacillus subtilis are able to take up single-stranded homologous donor DNA fragments in vitro to form donor-recipient DNA complexes (Van Randen and Venema 1981). The unfolded chromosomes behave as supercoiled DNA molecules. X-irradiation increased the formation of unstable and stable complexes between donor and recipient DNA during incubation at 37 degrees C. The complex-forming ability of the unfolded chromosomes increased linearly with increasing X-ray dose, even after complete relaxation of the unfolded chromosomes had occurred. Limited DNase I action increased the complex-forming ability of the chromosomes as effectively as X-irradiation. Unstable donor-recipient DNA complexes can be distinguished from stable ones by their dissociation upon density gradient centrifugation in CsCl at pH 11.2. They are stable at pH 10 (Van Randen et al. 1982a). At an intermediate pH value during isopycnic centrifugation, a fraction of the unstable complexes were stable, suggesting that a range of stabilities existed among the unstable complexes. The donor moiety of the stable donor-recipient DNA complexes was far more resistant to nuclease S1 treatment than that of the unstable ones.

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Unstable association in vitro between donor and recipient DNA in Bacillus subtilis

Cited by 3 publications

References 20 publications

Initiation of recombination during transformation of Bacillus subtilis requires no extensive homologous sequences

Initiation of recombination during transformation of Bacillus subtilis requires no extensive homologous sequences

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

Involvement of single-strand breaks in complex formation between single-stranded DNA and nucleoids of Bacillus subtilis

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