Abstract
Monofunctional alkylating agents preferentially react at the N7 position of 2'-deoxyguanosine in duplex DNA. Methylated DNA, such as that produced by methyl methanesulfonate (MMS) and temozolomide, exists for days in organisms. The predominant consequence of N7-methyl-2'-deoxyguanosine (MdG) is widely believed to be abasic site (AP) formation via hydrolysis, a process that is slow in free DNA. Examination of MdG reactivity within nucleosome core particles (NCPs) provided two general observations. MdG depurination rate constants are reduced in NCPs compared with when the identical DNA sequence is free in solution. The magnitude of the decrease correlates with proximity to the positively charged histone tails, and experiments in NCPs containing histone variants reveal that positively charged amino acids are responsible for the decreased rate of abasic site formation from MdG. In addition, the lysine-rich histone tails form DNA-protein cross-links (DPCs) with MdG. Cross-link formation is reversible and is ascribed to nucleophilic attack at the C8 position of MdG. DPC and retarded abasic site formation are observed in NCPs randomly damaged by MMS, indicating that these are general processes. Histone-MdG cross-links were also detected by mass spectrometry in chromatin isolated from V79 Chinese hamster lung cells treated with MMS. The formation of DPCs following damage by a monofunctional alkylating agent has not been reported previously. These observations reveal the possibility that such DPCs may contribute to the cytotoxicity of monofunctional alkylating agents, such as MMS, N-methyl-N-nitrosourea, and temozolomide.