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-N-nitrosoguanidine (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 the elucidation of 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, that is, 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 the damage is not repaired.

Figure 1

Potential sites of chemical methylation in double strand DNA. The arrows point to each methyl adduct and whether the adduct is known to be predominantly toxic or mutagenic. The open arrows represent sites that are methylated by MMS, MNNG, and MNU. The filled arrows point to sites that are methylated by MNNG and MNU, but not detectably by MMS. Note that methylation of different sites on the same base at the same time is extremely rare. The size of the arrows roughly represent the relative proportion of adducts. In single strand DNA, the N1-adenine and N3-cytosine positions display a greater reactivity.

Figure 2

Repair of methyl damage in E. coli. Arrows identify methyl groups or methyl bases removed by the specific proteins for each position. The arrows do not represent adduct proportions. Ogt, Ada, and AlkB directly remove the methyl group to reverse the damage. Tag and AlkA are DNA glycosylases that initiate BER. N7-MeA is a relatively rare adduct that depurinates rapidly and no repair mechanism is known.

Figure 3

Repair of methyl damage in mammalian cells. Arrows identify methyl groups or methyl bases removed by the specific proteins for each position. MGMT and ABH2 directly remove the methyl group to reverse the damage. AAG is a DNA glycosylase that initiates BER. Question marks are shown where adduct repair has not yet been definitively identified.

Figure 4

General schematic of base excision repair following methyl base damage. The first step is carried out by a DNA glycosylase, which produces an abasic site. The second step is carried out by an AP endonuclease, which creates a nick 5′- to the abasic site. DNA synthesis replaces a single nucleotide (short patch) or several nucleotides (long patch). The 5′-dRP group is removed by 5′-dRP lyase (short patch) or as part of an overhang by a ‘flap’ endonuclease (long patch). DNA ligase seals the nick to complete the pathway. Incomplete BER intermediates, in particular the 5′-dRP group, are thought to lead to downstream consequences such as chromosomal aberrations and toxicity.

Figure 5

General schematic of the recognition of an O⁶-MeG:T mispair by the proteins of mismatch repair. In the first step, the MSH2:MSH6 heterodimer (MutSα) recognizes O⁶-MeG:T. In the second step, MutSα is recognized by the MLH1:PMS2 heterodimer (MutLα). The downstream consequences of these events include the induction of DNA damage signaling events, G2/M cell cycle arrest, induction of sister chromatid exchanges and apoptosis.

Figure 6

Events downstream of methyl base damage can lead to homologous recombination repair. BER intermediates or unrepaired methyl damage encountered by replication, or MMR recognition of O⁶-MeG:T mispairs can induce homologous recombination events. Homologous recombination promotes strand invasion onto a sister chromatid template to initiate repair. DNA synthesis and resolution of the Holliday junction completes the process. If left unrepaired, double strand breaks lead to chromosomal rearrangements.