The role of transcription in repair of UV-induced CPD was investigated in the <I>Saccharomyces cerevisiae</I> <I>MFA2</I> gene. <I>MFA2</I> mutants were constructed for cloning into a yeast artificial chromosome (YAC) by deletion of either the 22nt TATA box sequences (T2Δ<I>MFA2</I>) or 55nt Mcm1 binding site (MΔ<I>MFA2</I>) from its promoter. <I>In vivo</I> transcription analysis using eGFP reporter system reveals a 10-fold reduction in transcription activity for the T2Δ<I>MFA2</I> a cells compared to the WT<I>MFA2 </I>a cells. This reduced transcription level is very close to the level found in α cells where <I>MFA2</I> is inactive. A 7-fold reduction in transcription activity was found for MΔ<I>MFA2</I> in both a and α cells. This result indicates the deletion of the MBS eliminates Mcm1/α2-mediated <I>MFA2</I> repression in α cells dramatically reduced <I>MFA2 </I>expression in a cells. Among 13 CPDs found in the promoter region, three CPDs are increasingly induced following the deletion of the TATA box and MBS sequences. However, their repair rates are not affected. Analysis of NER was performed on individual CPDs in both the transcribed (TS) and non-transcribed (NTS) strands by comparison between the YAC-borne <I>MFA2 </I>and the endogenous gene for each construct. The endogenous <I>MFA2</I> in YAC strains harbouring the WT<I>MFA2</I> and MΔ<I>MFA2</I> constructs, exhibit an identical repair profile. This suggests no genetic variation occurs within the isogenic strain harbouring the three promoter element deletion constructs. The repair results on the TS indicate the fastest rate with t<SUB>50%</SUB> of 1.5 hr for some CPDs in the transcription region of the transcribed strand of the WT<I>MFA2</I>.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:637808 |
| Date | January 2001 |
| Creators | Klungsupya, P. |
| Publisher | Swansea University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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