Base excision repair in a glucocorticoid response element: effect of glucocorticoid receptor binding.
DNA repair takes place in the context of chromatin. Previous studies showed that histones impair base excision repair (BER) of modified bases at both the excision and synthesis steps. We examined BER of uracil in a glucocorticoid response element (GRE) complexed with the glucocorticoid receptor DNA binding domain (GR-DBD). Five substrates were designed, each containing a unique C-->U substitution within the mouse mammary tumor virus promoter, one located within each GRE half-site and the others located outside the GRE. To examine distinct steps of BER, DNA cleavage by uracil-DNA glycosylase and Ape1 endonuclease was used to assess initiation, dCTP incorporation by DNA polymerase (pol) beta was used to measure repair synthesis, and DNA ligase I was used to seal the nick. For uracil sites within the GRE, there was a reduced rate of uracil-DNA glycosylase/Ape1 activity following GR-DBD binding. Cleavage in the right half-site, with higher GR-DBD binding affinity, was reduced approximately 5-fold, whereas cleavage in the left half-site was reduced approximately 3.8-fold. Conversely, uracil-directed cleavage outside the GRE was unaffected by GR-DBD binding. Surprisingly, there was no reduction in the rate of pol beta synthesis or DNA ligase activity on any of the fragments bound to GR-DBD. Indeed, we observed a small increase ( approximately 1.5-2.2-fold) in the rate of pol beta synthesis at uracil residues in both the GRE and one site six nucleotides downstream. These results highlight the potential for both positive and negative impacts of DNA-transcription factor binding on the rate of BER.
Hinz
Angela K
AK
Biochemistry and Biophysics, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-7520, USA.
Wang
Yan
Y
Smerdon
Michael J
MJ
D000818:Animals; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology*; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008324:Mammary Tumor Virus, Mouse / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D011965:Receptors, Glucocorticoid; D020218:Response Elements; D051981:Uracil-DNA Glycosidase / Q000737:chemistry / Q000378:metabolism
D004279:DNA, Viral; D011965:Receptors, Glucocorticoid; D019951:DNA Polymerase beta; D051981:Uracil-DNA Glycosidase; C085108:APEX1 protein, human; C486216:Apex1 protein, mouse; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase