TY - JOUR
T1 - Transfection enhancement in Bacillus subtilis displays features of a novel DNA repair pathway I
T2 - DNA base and nucleolytic specificity
AU - Radany, Eric H.
AU - Malanoski, Gregory
AU - Ambulos, Nicholas P.
AU - Friedberg, Errol C.
AU - Yasbin, Ronald E.
N1 - Funding Information:
We thank the several investigators noted for gifts of strains and enzymes, and Kenneth Dornfeld for thoughtful comments on the draft manuscript. This work was supported in part by grants from the N.I.H. (1K08-CA-1590) to E.H.R., and the National Science Foundation (MCB-9219436) to R.E.Y.
PY - 1997/8
Y1 - 1997/8
N2 - 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.
AB - 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.
KW - Bacillus subtilis
KW - DNA repair
KW - Transfection enhancement
KW - UV radiation
UR - http://www.scopus.com/inward/record.url?scp=0030789133&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030789133&partnerID=8YFLogxK
U2 - 10.1016/S0921-8777(97)00019-0
DO - 10.1016/S0921-8777(97)00019-0
M3 - Article
C2 - 9298119
AN - SCOPUS:0030789133
SN - 0921-8777
VL - 384
SP - 107
EP - 120
JO - Mutation Research - DNA Repair
JF - Mutation Research - DNA Repair
IS - 2
ER -