Methylating Agents and DNA Repair Responses:  Methylated Bases and Sources of Strand Breaks

Abstract

The chemical methylating agents methylmethane sulfonate (MMS) and N-methyl-N′-nitro-Nnitrosoguanidine (MNNG) have been used for decades as classical DNA damaging agents. These agents have been utilized to uncover and explore pathways of DNA repair, DNA damage response, and mutagenesis. MMS and MNNG modify DNA by adding methyl groups to a number of nucleophilic sites on the DNA bases, although MNNG produces a greater percentage of O-methyl adducts. There has been substantial progress elucidating direct reversal proteins that remove methyl groups and base excision repair (BER), which removes and replaces methylated bases. Direct reversal proteins and BER thus counteract the toxic, mutagenic and clastogenic effects of methylating agents. Despite recent progress, the complexity of DNA damage responses to methylating agents is still being discovered. In particular, there is growing understanding of pathways such as homologous recombination, lesion bypass, and mismatch repair that react when the response of direct reversal proteins and BER is insufficient. Furthermore, the importance of proper balance within the steps in BER has been uncovered with the knowledge that DNA structural intermediates during BER are deleterious. A number of issues complicate elucidating the downstream responses when direct reversal is insufficient or BER is imbalanced. These include inter-species differences, cell-type specific differences within mammals and between cancer cell lines, and the type of methyl damage or BER intermediate encountered. MMS also carries a misleading reputation of being a 'radiomimetic,' i.e., capable of directly producing strand breaks. This review focuses on the DNA methyl damage caused by MMS and MNNG for each site of potential methylation to summarize what is known about the repair of such damage and the downstream responses and consequences if not repaired.

DNA Damage Response and DNA Repair in Skeletal Myocytes From a Mouse Model of Spinal Muscular Atrophy

“…In the "2B/-"model, Pax7 and MyoD expression levels were reduced in SMA myoblasts before differentiation (36). The "2B/-"mice are maintained on a hybrid C57BL/6xCD1 background (83). The CD1 mouse strain is known to differ from other mouse strains on physiological and genetic measures (72)(73)(74)(75).…”

“…The mechanism of action of MMS is direct-acting DNA base alkylation (82). The major MMS-induced base lesions are 7-methylguanine and 3-methyladenine (83). The downstream effects of MMS are complex.…”

“…MMS has radiomimetic properties with the induction of DNA-SSBs and -DSBs; however, it is not clear whether it directly causes DNA strand breaks or whether the strand breaks are the result of repair intermediates or heatlabile DNA damage at replication forks (82). The repair of MMS-induced DNA damage is through direct reversal repair of methylated DNA bases and by base excision repair that removes and replaces methylated bases (83). The findings that IR-and etoposide-induced DNA damage is repaired successfully by differentiated myotubes, but not DNA damaged caused by MMS, indicate that direct reversal repair of DNA is underrepresented in differentiated skeletal muscle.…”

The role of Cockayne Syndrome group B (CSB) protein in base excision repair and aging

“…MMS introduces BER substrates/intermediates. The most critical biological lesion caused by MMS is presumed to be N-methylation base products, which frequently give rise to AP sites via enhanced hydrolysis of the N-glycosylic bond or DNA glycosylase-mediated base release (Wyatt and Pittman, 2006). CSB deficient cells expressing an ATPase domain II mutant CSB protein (CSBE646Q) display intermediate sensitivity to MMS challenge.…”

Control of Trx1 redox state modulates protection against methyl methanesulfonate-induced DNA damage via stabilization of p21

“…MMS also has a misleading reputation as a radiomimetic capable of directly producing DNAstrand breaks (1). Studies showed that MMS treatment increased intracellular reactive oxygen species (ROS) in Saccharomyces cerevisiae (2,3), and may further depleted glutathione and induced lipid peroxidation and cell death, which processes were reversed by antioxidant activity (4).…”