Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway II: host constitutive expression, repair DNA synthesis, and in vitro activity

Eric H. Radany, Gregory Malanoski, Nicholas P. Ambulos, Errol C. Friedberg, Ronald E. Yasbin

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

In the Bacillus subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, sometimes followed by productive cell infection. Previous studies have shown that ultraviolet (UV)-irradiation of the competent host cells, or cotransfection of UV-irradiated heterologous DNA, can increase the efficiency of transfection in some cases; these latter two phenomena have been called transfection enhancement (TE). In an accompanying paper, we show that the TE is apparently confined to the B. subtilis phages that contain hydroxymethyluracil (HMU) in their DNA, and that the photoproduct in UV-irradiated DNA that mediates TE is specific, and differerent than the pyrimidine dimer, thymine glycol, uracil, or HMU. We also show that TE is due to reduced intracellular endonucleolytic attack of transfecting DNA. Based on this DNA base and nucleolytic specificity, we hypothesized that TE reflects the incidental action of a host DNA repair system on transfecting HMU phage DNA. In continuing these studies, we show here that duplex infecting HMU phage DNA is apparently inactivated by this same putative repair system when phage protein synthesis is blocked. We find, too, that this inactivation of infecting HMU phage DNA can be inhibited by UV-irradiated DNA, and that this process has a similar DNA base specificity as for TE. The survival of infecting HMU phage DNA is dependent on host DNA polymerase activity. We can detect specific DNA synthesis consist with formation of repair patches when inactivation of infecting HMU phase DNA is ongoing, but not when it is inhibited by the presence of UV DNA or by allowing phage gene expression. Each of these results is consistent with the hypothesis that TE reflects the action of a novel DNA repair pathway. We show that a candidate TE-associated enzymatic activity can be detected in cell free extracts of uninfected, but not HMU phage-infected B. subtilis cells. Correspondingly, the extracts of phage-infected cells appear to contain a diffusible factor that acts as an inhibitor of this host enzyme.

Original languageEnglish (US)
Pages (from-to)121-134
Number of pages14
JournalMutation Research - DNA Repair
Volume384
Issue number2
DOIs
StatePublished - Aug 1997

Fingerprint

Bacilli
Bacillus subtilis
DNA Repair
Transfection
Repair
Bacteriophages
DNA
In Vitro Techniques
Bacillus Phages
Pyrimidine Dimers
Uracil
5-hydroxymethyluracil
Enzyme Inhibitors
DNA-Directed DNA Polymerase
Cell Extracts
Gene expression

Keywords

  • Bacillus subtilis
  • DNA repair
  • Transfection enhancement
  • UV irradiation

ASJC Scopus subject areas

  • Toxicology
  • Genetics
  • Molecular Biology

Cite this

Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway II : host constitutive expression, repair DNA synthesis, and in vitro activity. / Radany, Eric H.; Malanoski, Gregory; Ambulos, Nicholas P.; Friedberg, Errol C.; Yasbin, Ronald E.

In: Mutation Research - DNA Repair, Vol. 384, No. 2, 08.1997, p. 121-134.

Research output: Contribution to journalArticle

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