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Binary system for selective photoaffinity labeling of base excision repair DNA polymerases
Olga I Lavrik1, Dmitry M Kolpashchikov, Rajendra Prasad
1Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T. W. Alexander Drive, Research Triangle Park, NC 27709, USA.
Nucleic Acids Research
|July 24, 2002
Summary
Researchers developed a new method to selectively label DNA polymerases in cellular extracts. This technique uses photoaffinity reagents to identify DNA polymerase beta as the primary enzyme crosslinked in mouse embryonic fibroblast extracts.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Selective labeling of DNA polymerases is crucial for understanding DNA repair and replication.
- Existing methods may lack specificity or require purified enzyme preparations.
- Crude cellular extracts present challenges for targeted protein identification.
Purpose of the Study:
- To develop and validate a novel photoaffinity labeling system for selective identification of DNA polymerases in crude cellular extracts.
- To investigate the utility of a pyrene-activated arylazido photoreactive group for in situ labeling.
- To identify the predominant DNA polymerase crosslinked in mouse embryonic fibroblast (MEF) extracts.
Main Methods:
- In situ synthesis of a photoreactive long patch base excision repair (BER) intermediate using DNA substrates with abasic sites and base-substituted arylazido-dUTPs in MEF extracts.
- Activation of the arylazido group via energy transfer from a pyrene group (Pyr-dUTP) upon 365 nm UV light exposure.
- Analysis of crosslinked proteins by gel electrophoresis and identification of labeled DNA polymerases.
Main Results:
- The developed system selectively crosslinked DNA-bound DNA polymerases to the photoreactive BER intermediate.
- DNA polymerase beta was identified as the predominant crosslinked protein in MEF extracts.
- Non-specific DNA binding proteins were not crosslinked under these specific activation conditions.
- Direct UV activation at 312 nm resulted in labeling of several other DNA binding proteins, indicating method specificity.
Conclusions:
- A novel and selective photoaffinity labeling method has been established for identifying DNA polymerases in crude cellular extracts.
- The pyrene-activated system demonstrates high specificity for targeting DNA polymerases, particularly DNA polymerase beta.
- This methodology offers a valuable tool for studying DNA polymerases in complex biological samples without prior purification.