Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway. II: Host constitutive expression, repair DNA synthesis, and in vitro activity.
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 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 different 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 consistent with formation of repair patches when inactivation of infecting HMU phage 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.
Radany
E H
EH
Department of Radiation Oncology, University of Michigan School of Medicine, Ann Arbor 48109-0582, USA. Eric_Radany@mailgw.surg.med.umich.edu
Malanoski
G
G
Ambulos
N P
NP
Friedberg
E C
EC
Yasbin
R E
RE
D001412:Bacillus subtilis / Q000235:genetics* / Q000528:radiation effects / Q000821:virology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000528:radiation effects; D001435:Bacteriophages / Q000235:genetics / Q000528:radiation effects; D004249:DNA Damage / Q000235:genetics / Q000528:radiation effects; D004260:DNA Repair / Q000235:genetics* / Q000528:radiation effects; D004269:DNA, Bacterial / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects*; D004279:DNA, Viral / Q000096:biosynthesis / Q000528:radiation effects; D004259:DNA-Directed DNA Polymerase / Q000187:drug effects / Q000235:genetics; D010432:Pentoxyl / Q000031:analogs & derivatives / Q000378:metabolism / Q000528:radiation effects; D011092:Polyethylene Glycols / Q000494:pharmacology; D013379:Substrate Specificity; D014162:Transfection / Q000379:methods; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004279:DNA, Viral; D011092:Polyethylene Glycols; C008055:5-hydroxymethyluracil; D010432:Pentoxyl; D004259:DNA-Directed DNA Polymerase