Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway. I: DNA base and nucleolytic specificity.
Cells of Bacillus subtilis can enter a natural physiological state, termed competence, that is permissive for uptake of DNA from the surrounding medium. In the B. subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, followed by intracellular processing that may ultimately lead to productive infection. Previous investigations have shown that transfecting DNA is usually far less infectious (on a molar basis) than is the DNA injected by phage particles; this result is apparently due to inactivating events suffered by transfecting DNA during its metabolism by competent cells. Earlier studies also demonstrated that, in some cases, the infectivity of transfecting DNA can be increased by ultraviolet (UV) irradiation of the competent cells prior to transfection, or by cotransfection of UV-irradiated heterologous DNAs; collectively, these phenomena have been termed transfection enhancement (TE). We propose here that some transfecting B. subtilis phage DNAs are attacked by a novel host DNA repair system, and that TE reflects inhibition of this by a competing substrate in UV-irradiated DNA. In support of this model, we show that UV-DNA cotransfection leads to a reduced rate of intracellular endonucleolytic breakdown of transfecting DNA. We also demonstrate that TE displays marked specificity of a kind frequently observed for repair enzymes. Thus, phages that contain hydroxymethyl uracil (HMU), but not thymine, in their genomes are susceptible to this process. In addition, we show that the photoproduct(s) in UV-irradiated DNA that produces TE by cotransfection is specific, and is not uracil, a pyrimidine dimer, thymine glycol, HMU, or a substrate for the E. coli thymine glycol DNA N-glycosylase. This photoproduct is derivable from thymine or HMU. The implications of these results are discussed.
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; D001435:Bacteriophages / Q000235:genetics / Q000528:radiation effects; D001482:Base Composition; D045647:DNA Glycosylases; D004260:DNA Repair / Q000235:genetics* / Q000528:radiation effects; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects*; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D010432:Pentoxyl / Q000031:analogs & derivatives / Q000378:metabolism / Q000528:radiation effects; D011743:Pyrimidines / Q000378:metabolism / Q000528:radiation effects; D013379:Substrate Specificity; D013941:Thymine / Q000031:analogs & derivatives / Q000378:metabolism / Q000528:radiation effects; D014162:Transfection / Q000379:methods*; D014466:Ultraviolet Rays; D051981:Uracil-DNA Glycosidase
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011743:Pyrimidines; C029389:thymine glycol; C008055:5-hydroxymethyluracil; D010432:Pentoxyl; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; C030986:pyrimidine; D013941:Thymine