TRANSFECTION ENHANCEMENT IN BACILLUS-SUBTILIS DISPLAYS FEATURES OF A NOVEL DNA-REPAIR PATHWAY .2. 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 foll owed by productive cell infection. Previous studies have shown that ul traviolet (UV)-irradiation of the competent host cells, or cotransfect ion of W-irradiated heterologous DNA, can increase the efficiency of t ransfection 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 hydr oxymethyluracil (HMU) in their DNA, and that the photoproduct in UV-ir radiated DNA that mediates TE is specific, and different than the pyri midine dimer, thymine glycol, uracil, or HMU. We also show that TE is due to reduced intracellular endonucleolytic attack of transfecting DN A. 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 D NA 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 inactiv ation of infecting HMU phage DNA is ongoing, but not when it is inhibi ted by the presence of UV DNA or by allowing phage gene expression. Ea ch 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 o f uninfected, but not HMU phage-infefted, B. subtilis cells. Correspon dingly, the extracts of phage-infected cells appear to contain a diffu sible factor that acts as an inhibitor of this host enzyme. (C) 1997 E lsevier Science B.V.