DNA in proximity to the site of replication is preferentially alkylated in S phase 10T1/2 cells treated with N-methyl-N-nitrosourea

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Abstract
Replicating DNA is more susceptible to modification by N-methyl-N-nitrosourea (MNU), a spontaneously active methylating agent, than bulk DNA. This conclusion is supported by results from two different experimental approaches. First, synchronized C3H 10T1/2 clone 8 cells were treated in S phase with MNU and DNA replicated during the period of treatment was separated from bulk DNA. This was done by digesting the purified DNA with restriction enzymes and retaining the replication fork-associated DNA in nitrocellulose filters. Second, synchronized C3H 10T1/2 clone 8 cells were exposed to 5-bromodeoxyuridine and [3H]MNU and the density-labelled, replicated DNA was separated in CsCl gradients. Both methods show 2.6 to 5.0 times more [3H]methyl adducts per nucleotide residue associated with replicating DNA than that expected from random methylation. These experiments were done at low MNU concentrations (0.018 – 0.115 mM) that did not cause any detectable inhibition of DNA synthesis or stimulation of repair replication in 10T1/2 cells.