Genetic Analysis of Repair of Ultraviolet Damage by Competent and Noncompetent Cells of Bacillus subtilis

Abstract

The repair of ultraviolet (UV) damage in Bacillus subtilis W23T − has been studied by transformation with deoxyribonucleic acid (DNA) extracted from irradiated cells before and after repair. The extent of repair of genetic markers by donor cells after low or moderate doses of UV was found to be related only to the initial degree of inactivation. After a very high dose, further inactivation occurred, also in proportion to initial damage. In addition, the competent recipient cells were shown to repair approximately 75% of the damage in transforming DNA. The sensitivities of markers irradiated either in vivo or in vitro appeared to be related to map position, the more proximal markers showing a greater resistance to UV inactivation.

Repair and subsequent fragmentation of deoxyribonucleic acid in ultraviolet-irradiated Bacillus subtilis recA

“…UJnless otherwise noted, cells were grown at 37°C in mo(lified (3) Spizizen salts containing 0).5'r.c glucose, 0.01'i acid-hydrolyze(d casein (CAA, I)ifco, Detroit, Mich.), anti ( 5)) pg each of niethioriniie and(l trvptophan per ml. P'reparation oftconpetenit cells and transformiationi were carried out as describedl p)reviously (12). For labeling of DNA with 'H, 2'-deoxyadenosine (30jitg/ml) and [HIdThd (20 pCi/ml) were added to the growth medium.…”

“…Irradiation. Bacteria and transforming DNA were irradiated with UV as described previously (12). Xirradiation was done with a G. E. Maxitron 250 at a peak voltage of 250 kV and a current of 30 mA, with a 3-mm aluminum filter.…”

Postreplication repair of ultraviolet-irradiated transforming deoxyribonucleic acid in Bacillus subtilis

“…Samples of 0.6 ml at a concentration of 100 pg/ml were irradiated in a layer about 1 mm deep while being stirred in a watch glass. Preparation of competent cells and transformation were done as previously described (20). Recombinant DNA (see Tables 2 and 3) was extracted by diluting 6 ml of the transformed culture with 6 ml of ice-cold buffer containing 40 mM Tris and 60 mM EDTA at pH 8.1.…”