M ol Gen Genet (1983) 189 : t 18-122 MGG © Springer-Verlag 1983 Repair of Methyl Methane Sulfonate-Damaged Phage by Haemophilus intluenzae Johan H. Stuy and Hasan Bagci* Department of Biological Science, Florida State University, Tallahassee, Florida 32306, USA Summary. Seven mutants of Haemophilus influenzae strain Rd (mmsA-) have been isolated that are more sensitive to methyl methane sulfonate (mms) than recombination- deficient (recA) mutants. The mutations cotransformed about 25% with the strA locus while the five studied clus- tered tightly; they are all probably allelic. The mutants are not sensitive to ultraviolet radiation, X-rays, or nitrous acid. Mms-damaged phage HP1 plated very inefficiently on these mutants, indicating that they lack the first step in the excision repair of the lesion N3-methyladenine (m3A). Incubation of damaged phage at 30°C in the absence of mms resulted in a steady decline of viability when the phage were plated on the wild mmsA ÷ host but an initial steep rise was seen when it was plated on an mmsA- mutant. The rise is explained by the assumption that m3A lesions hydrolyzed off the DNA giving rise to repairable apurinic sites by both the mmsA + and mmsA- hosts. No decline in viability was observed when hydroxyl- amine was present in the medium. This compound is known to prevent or slow down t-elimination. The delayed decline in viability is therefore explained by assuming that apurinic sites give rise to//-elimination-induced single strand breaks in the phage DNA that cannot be repaired by either host. Marker rescue experiments indicated that these breaks did not interrupt injection of phage DNA. Introduction Treatment of DNA with methylating agents results in the formation of N7-methylguanine (mTG), N3-methyladenine (m3A), O6-methylguanine, and certain other minor com- pounds (Brookes and Lawley 1963 ; for a review, see Strauss et al. 1975). Methylated bases readily hydrolyze off the DNA (Zoltewicz et al. 1969) giving rise to apurinic sites. Apurinic sites, through deoxyribose linearization and subse- quent//-elimination, can cause single DNA strand breaks (Bayley et al. 1961 ; Jones et al. 1968). Strauss et al. (1975) have discussed calculations about how many methyl groups are fixed in DNA if an organism is exposed to methylating agents at 37 ° C, how fast the methylated purines are hydro- * Present address." Biology Department, Middle East Technical University, Ankara, Turkey Offprint requests to: J.H. Stuy lyzed off the DNA, and how fast the resulting apurinic sites give rise to single strand breaks (see also Lindahl and Andersson 1972). Eseherichia coli bacteria have a constitutive enzyme that removes m3A from DNA (m3A DNA glycosylase; Karran et al. 1980; Riazuddin and Lindahl 1978), and it is believed that all cells have such a glycosylase (Cathcart and Goldth- wait 1978; Brent 1979). The enzyme has a narrow substrate specificity (Karran et al. 1982). Mutants that do not have this enzyme (tag) show lack of host cell reactivation of mms-treated phage. It was recently shown that a second m3A DNA glycosylase is induced in E. coli by pretreatment with low concentrations of methylating agents (Karran et al. 1982; Evensen and Seeberg 1982). This glycosylase appears also to remove raTA as well as mTG. When alkylated phage was incubated in the absence of the alkylating agent, a steady decline in viability was ob- served (Brakier and Verly 1970; Lawley et al. 1969; Love- less 1959). There is general agreement that this decline is caused by the hydrolysis of the methylated purines, which gives rise to apurinic sites. We have isolated seven mutants of Haemophilus influen- zae that are more sensitive to mms than the recombination- deficient recA1 mutant (rec-1; Setlow et al. 1968). These seven mutants Showed lack of host cell reactivation of phage HP1. Incubation of treated phage caused a steady decline in viability when the phage was titrated on a wild host, but an initial steep rise was seen when an mmsA- host was used. The decline was not seen when hydoxylamine was present in the incubation medium. We therefore believe that the delayed inactivation is caused by t-elimination- induced single DNA strand breaks. Materials and Methods Strains. All H. influenzae strains described were derivatives of the BC200 strain (Barnhart and Cox 1968). Phage HPI was obtained from C. Rupert (Harm and Rupert 1963). The recA1 mutation was given to us by J. Setlow (her ree-1 or DB117; Setlow et al. 1968). Media. Broth was 3% Difco brain heart infusion supple- mented with 10 lag/ml of hemin and of nicotinic acid amide adenine dinucleotide. The pH was lowered to 7 by adding 0.5% (v/v) N HC1. Hard agar contained 1.3% agar; soft agar contained 0.7% agar. 0026-8925/83/0189/0118/$01.00