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Molecular regulatory mechanisms of DNA damage-inducible genes, MAG1 and DDI1, from saccharomyces cerevisiae



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My research project involved dissecting cis-acting promoter elements and attempting to identify binding proteins that regulate the expression and mediatc the DNA damage response of the yeast genes MAG1 and DDI1. MAG1 encodes a 3-methyladenine (3MeA) DNA glycosylase and protects cells against killing by MMS-induced DNA replication blocks (Chen et al., 1989 Proc. Natl. Acad. Sci. USA 86: 7961-7965). DDI1 was recently identified as a gene upstream of MAG1 and was inducible by DNA damaging agents (Xiao and Fontaine, unpublished). MAG1 and DDI1 are arranged in a head-to-head configuration and are transcribed divergently. These two genes are closely linked, with the fust ATG's of the two open reading frames being separated by 282 base pairs. The transcription of MAG1 is repressed by a URS (upstream repressing site) element and stimulated in response to DNA damage by a putative UAS (upstream activating site) (Xiao et al., 1993 Mol. Cell. Biol. 13: 7213-7221). The 46 bp sequence containing the putative UAS of MAG1 (UASMAG1) is located within the coding region of DDI1. The transcriptional and the translational starts of MAG1 and DDI1 were determined. My results showed that the two genes are indeed closely linked to each other. The UASMAG1 was identified within the protein coding region of DDI1. This is the first demonstration in yeast that a transcriptional regulatory element for one gene can be located within the protein coding region of another gene. Since MAG1 and DDI1 are co-induced by DNA damage in a similar manner, it was hypothesized that the two genes share one or more regulatory elements. A direct repeat sequence (DR) within the intergenic region between MACI andDDII was identified as a bi-directional transcriptional regulatory element for the expression of these two genes. Sequences similar to the direct repeat were also found in the promoters of several DNA repair, or DNA metabolism genes from S. cerevisiae. This is the first report of a situation where two DNA damage-inducible genes are co-ordinately regulated by physically sharing a regulatory element. MAG1 is one of the most extensively studied yeast DNA damage responsive genes. Previous studies (Xiao et al., 1993 Mol. Cell. Biol. 13: 7213-7221) have focused primarily on the mechanism of repression of MAG1 expression. The UASMAG1 element was not well defined and its role in the induction of MAG1 following DNA damage was not established. This work defined the UASMAG1 as a 24 bp sequence required for the expression of MAG1, but not DDI1. An UASMAG1-binding protein(s) was identified. The UASMAG1-binding protein(s) is probably a transcription activator that regulates the expression of MAG1. (Abstract shortened by UMI.)



microbiology, molecular biology, genetics



Doctor of Philosophy (Ph.D.)


Microbiology and Immunology


Microbiology and Immunology




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