DNA-DNA and DNA-protein crosslinking and repair in Neurospora crassa following exposure to nitrogen mustard

A new method was developed for the analysis of DNA-protein crosslinks in Neurospora crassa. The formations of DNA-protein and DNA-DNA crosslinks were assayed following exposure of spheroplasts to HN2. Both types of crosslink were detected and were found to be repaired during recovery. Moreover a mutant sensitive to HN2 was defective in the removal of both types of crosslink.     

Inactivation of bacteriophage λ and λ DNA by nitrogen mustard

Bacteriophage λ and λ DNA were treated with alkylating agents. The survival of phage was assayed by infectivity and that of DNA by infectivity of phage particles assembled from the DNA in vitro. Phage λ were more sensitive to nitrogen mustard (Cl(CH₂)₂NMe(CH₂)₂Cl; HN2) than was λ DNA. The inactivation of λ DNA was biphasic; the second component of the inactivation was sensitive to mutations allelic for recA, polA and uvrB. This behaviour was not shown by pBR322 plasmid DNA treated with HN2 nor by λ DNA treated with monofunctional alkylating agents (or HN2 if the second alkylation reaction was stopped by addition of a mercaptan). From Arrhenius plots, the activation energy for the reactions with DNA and intact phage were found to be different. The activation energy for the inactivation of intact phage was the same as that (measured independently) for the predominant reaction (or class of reactions) in which HN2 cross-links DNA to protein in λ particles. From these data we conclude that the inactivation of λ by HN2 is due, primarily, to DNA-protein cross-linking. The implications for the mode of action of DNA-reactive bifunctional anti-viral and cytotoxic compounds are discussed.     

Stabilities of 7-alkylguanosines and 7-deoxyguanosines formed by phosphoramide mustard and nitrogen mustard

7-Alkylguanosine and 7-alkyldeoxyguanosine were prepared from phosphoramide mustard and nitrogen mustard in nonaqueous conditions. The guanosine products were N-(2-chloroethyl)-N-[2-(7-guanosinyl)ethyl] phosphorodiamidic acid, and N-(2-chloroethyl)-N-[2-(7-guanosinyl)ethyl]methylamine, respectively. These were also formed in aqueous reactions but they rapidly underwent secondary reactions. The half-life of the phosphoramide mustard-guanosine adduct was 3.1 h (37 degrees C, pH 7.4) and that of the nitrogen mustard adduct 1 h (25 degrees C, pH 7.4), as determined by HPLC. The half-lives of the respective adducts for imidazole ring-opening were 9.5 h and 0.78 h (37 degrees C, pH 7.4). The respective deoxyguanosine derivatives depurinated with half-lives of 8.5 h and 1.6 h (25 degrees C, pH 4.2). The mustard adducts are notably more labile than simple alkyl substituted guanosines and deoxyguanosines.