Transcriptional response of O⁶-methylguanine methyltransferase deficient yeast to methyl-N-nitro-N-nitrosoguanidine (MNNG)

Abstract

(cont.) of transcription factors and subsequently, induction of RNA processing (35% of genes incrementally induced) and kinases involved in protein phosphorylation. In the WT, the response was restricted to a transient repression of fundamental biochemical processes. Interestingly, a gene whose repression is known to mimic apoptosis was found to be repressed in the WT. The overwhelming induction of ribosomal protein synthesis genes in both WT and mgtl in response to MNNG is an unexpected result that could signify a successful recovery following wide-spread cellular damage.

Damage to DNA can occur by means of endogenous biochemical processes or exogenous chemicals such as alkylating agents. If left unrepaired, alkylated bases, most notably, O⁶ Methylguanine (O⁶MeG) can be mutagenic and cytotoxic to the cell. Luckily, DNA methyltransferase (encoded by the gene MGT1 in yeast), repairs this damage. By using transcriptional profiling as a tool, an attempt to elucidate the role of MGT1 has been made. First, the basal expression profile of the mgtl was established. Then, the response of wild-type (WI) yeast and yeast lacking MGT1 (mgt1) to the alkylating agent, MNNG was studied using exponentially growing WT and mgti cultures which were exposed to 30[mu]g/ml of MNNG for 10 to 60 minutes. Basal expression profile of yeast lacking MGT1 showed up-regulation of RETV7, a gene implicated in spontaneous mutagenesis. Response to MNNG was invoked immediately and was dramatic and widespread involving 30% of the genome in both WT and mgt1. Cell-cycle checkpoints, damage signal amplifiers, DNA repair genes (nucleotide excision repair, photoreactive repair, mismatch repair) and chromatin remodeling genes were induced. Genes involved in maintaining mitochondrial structure and mitochondrial genome were also induced. Intriguingly, RPN4, a key regulator of proteasomal system was found to be repressed. Environmental stress response genes were culled out to examine the effects of MNNG on WT and mgtl, more carefully. Temporal gene expression profiles in WT and mgtl were informative in delineating differences in the distinct responses mounted by WT and mgtl. The magnitude of response in mgt1 is more profound than in WT. The differences in the dynamic trends between the two suggest that mgt1 initiates a coordinated response involving repression