Chimeric Proteins to Detect DNA Damage and Mismatches
The goal of this project was to develop chimeric proteins composed of a DNA mismatch or damage binding protein and a nuclease, as well as methods to detect DNA mismatches and damage. We accomplished this through protein engineering based on using polymerase chain reactions (PCRs) to create chimeras with novel functions for damage and mismatch detection. This project addressed fundamental questions relating to disease susceptibility and radiation-induced damage in cells. It also supported and enhanced LLNL's competency in the emerging field of proteomics. In nature, DNA is constantly being subjected to damaging agents such as exposure to ultraviolet (UV) radiation and various environmental and dietary carcinogens. If DNA damage is not repaired however, mutations in DNA result that can eventually manifest in cancer and other diseases. In addition to damage-induced DNA mutations, single nucleotide polymorphisms (SNPs), which are variations in the genetic sequence between individuals, may predispose some to disease. As a result of the Human Genome Project, the integrity of a person's DNA can now be monitored. Therefore, methods to detect DNA damage, mutations, and SNPs are useful not only in basic research but also in the health and biotechnology industries. Current methods of detection often use radioactive labeling and rely on expensive instrumentation that is not readily available in many research settings. Our methods to detect DNA damage and mismatches employ simple gel electrophoresis and flow cytometry, thereby alleviating the need for radioactive labeling and expensive equipment. In FY2001, we explored SNP detection by developing methods based on the ability of the chimeric proteins to detect mismatches. Using multiplex assays with flow cytometry and fluorescent beads to which the DNA substrates where attached, we showed that several of the chimeras possess greater affinity for damaged and mismatched DNA than for native DNA. This affinity was demonstrated in assays in which we looked both at (1) preferential degradation of damaged or mismatched DNA after addition of a chimeric protein, and (2) preferential binding of the chimeric proteins to damaged or mismatched DNA. In our experiments, we used various samples containing damage or mismatches and a control sample containing no damage or mismatches. When a chimeric protein was added to the DNA samples, the damage- or mismatch-recognition portion of the chimeric protein bound the samples containing the DNA damage or mismatch, allowing the nuclease portion to degrade the DNA. Thus, we measured both the preferential binding to the damaged or mismatched DNA and the preferential degradation of these same samples. We showed this in gel-based assays as well as in multiplex flow-cytometry assays. This year, we also focused on a thermophilic protein, MutS, which in nature functions to detect mistakes in DNA replication by its mismatch-detection capability in an organism that lives under extreme thermal conditions (75 C). Because of its thermophilic stability, this protein has great potential as a SNP detection tool. However, the results of our multiplex experiments were unclear-potentially because of interference between samples on different beads. In summary, our results using chimeric proteins and MutS suggest that the proteins and methods developed in this project have application for detecting SNPs and DNA damage as well as for genetic testing.
- Research Organization:
- Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- W-7405-ENG-48
- OSTI ID:
- 15004027
- Report Number(s):
- UCRL-ID-146788; TRN: US201015%%272
- Country of Publication:
- United States
- Language:
- English
Similar Records
Mutation detection by mismatch binding protein, MutS, in amplified DNA: Application to the cystic fibrosis gene
SBE primer : multiplexing minisequencing-based genotyping