Differences of DNA-Transcription Factor Interactions Following Chromium Exposure
The effect of 8-oxoguanine in the consensus sequence of êB DNA on the binding affinity of p50BD was determined using a 22-mer and a 30-mer double-stranded DNA oligonucleotide containing the NF-êB binding site. The addition of a carboxyl moiety at position 8 on guanine (creating 8-oxoguanine) was observed to change the binding affinity of the transcription factor for the êB DNA sequences at each modified guanine site. The protocol used a p50 binding domain protein (residues 23 to 366 from the p50 protein) and a p50 mutant protein with a single amino acid mutation of the wild type histidine 67 to alanine 67. Both proteins were cloned as N-terminal his6-tagged proteins. These proteins were expressed and purified as active proteins. It is known that reduction of chromium(VI) to chromium(III) creates species such as chromium(V) and chromium(IV) which interact with and damage DNA. An oxidative event, such a chromium(VI) reduction to chromium(III) could allow a crosslinking of protein-bound DNA. Redox values suggest that oxidative damage can travel to guanine-rich sites, such as that of the consensus sequence of êB DNA. This suggested a mechanism by which a chromium oxidative event could crosslink the DNA and protein into a binary complex without direct chromium involvement at the site of oxidation. Furthermore the protein, tightly bound to DNA, may compete with water to act as a nucleophile on DNA. This binary DNA-protein complex may be covalently linked and resistant to harsh conditions, allowing it to be visualized by HPLC or gel electrophoretic mobility shift assays. Under our conditions, no êB DNA, either unmodified or modified with 8-oxoguanine, was found to be bound to the p50BD protein. DNA oxidation through chromium reduction was not found to result in crosslinked p50BD protein in a binary DNAprotein complex.
Advisor:Dr. Kent Sugden; Dr. Michele McGuirl; Dr. Brooke Martin
School:The University of Montana
School Location:USA - Montana
Source Type:Master's Thesis
Date of Publication:12/05/2006