Host Cell Deoxyribonucleic Acid Polymerase I and the Support of T4 Bacteriophage Growth

Donna George, David Rosenberg
1972 Journal of Bacteriology  
Ultraviolet irradiation of Escherichia coli polAcells reduces their capacity to support the growth of T4 phage. There is no additional loss of capacity observed in pol tsA-recAdouble mutants at the nonpermissive temperature. The reversion frequency of a T4 rII mutant after ultraviolet irradiation is not changed by the absence of host deoxyribonucleic acid polymerase I. The in vivo function of deoxyribonucleic acid (DNA) polymerase I is not known; however, the in vitro properties of the enzyme
more » ... ies of the enzyme indicate a role in repair processes (5). A mutant of Escherichia coli strain W3110, polAl, containing less than 1% DNA polymerase I activity in vitro, is about five times as sensitive to ultraviolet light (UV) as the parental strain (4), although it is able to excise dimers normally (3). The polAl mutation also affects the survival of some, but not all, UV-sensitive on polAl than on polA+ cells (7), strain. Although OX174 and A phages are more UV-sensitive on polAI than on polA + cells (7) , the polymerase deficiency does not affect the survival of UV-irradiated T7 phage (4). We report here some observations on the effects of the polA1 mutation of the host on the survival and mutagenesis of UV-irradiated bacteriophage T4. Survival of phage after irradiation. T4 at a titer of 2 x 107/ml were suspended in tris(hydroxymethyl)aminomethane (Tris) diluent, placed on a rotating turntable to allow even exposure to all particles, and UV-irradiated with a 15-watt General Electric germicidal lamp (model no. G15T8) at a distance of 59 cm from the source. Under these conditions, an exposure of 1 sec corresponds to approximately 8 ergs/mm 2. Irradiated T4 phage were adsorbed to polAl and polA+ cells at 37C at a multiplicity of infection of 0.1. Unadsorbed phage were removed with anti-T4 serum. The survival of irradiated wild-type T4 is lower when assayed on polAl than on polA+ cells (Fig. 1) . After 40 sec of UV irradiation of the phage, the ratio of phage survival, polAl polA+, is 1:4.5. These results confirm the findings of Smith, Symonds, and White (9). However, the UV-sensitive mutant T4v and 101, wild-type phage T6 have the same UV sensitivity and plating efficiency on polA1 and polA+ cells (unpublished observations). T4v lacks an endonucleolytic activity (nicking enzyme) specific for UV-irradiated DNA (12) which apparently performs the first step in the excision repair of UV-induced lesions. Since the polAl mutation of the host does not further increase the UV sensitivity of T4v, it is probable that the effect of the polAl mutation on phage survival requires the presence of a single-stranded nick in the bacteriophage DNA. Reversion frequency of rll mutants. To determine whether the reduced survival of UV-irradiated T4+ on polAl cells is accompanied by an increased rate of mutagenesis, we assayed the reversion frequency of T4 r375 (an rII nonsense mutant revertable by T4 gene 43 mutator polymerase [10]) infecting polAl and polA+ cells. There were no significant differences in the reversion frequency of T4 r375 infecting polAl or polA+ cells, either before or after irradiation of the phage. The relative reversion frequency per surviving phage particle (on polAl/on polA+) ranged from 0.8 to 1.2 for unirradiated phage and from 0.9 to 1.0 for irradiated phage. Growth of T4+ on UV-irradiated cells. The capacity of E. coli to support the growth of T-even phages is normally very resistant to UV irradiation (2, 11). We investigated the effect of the polAl mutation on the capacity of UVirradiated cells to yield T4+ phage. Bacterial cells grown to 2 x 108/ml in M9S medium (1) were resuspended in Tris diluent at 1 x 109/ml. Irradiated cell suspensions were less than 1.0 mm in depth. The cells were exposed to increasing doses of UV and then infected at a multiplicity of 0.1 with wild-type T4. The phage, on May 9, 2020 by guest
doi:10.1128/jb.112.2.1017-1019.1972 fatcat:6xxaekyrlngbrmpv3qc6wcidrm