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Two forms of UvrC protein with different double-stranded DNA binding affinities
1Department of Environmental Medicine, New York University School of Medicine, Tuxedo, New York 10987.
The Journal of Biological Chemistry
|November 1, 2000
Summary
Researchers discovered two forms of UvrC protein, UvrCI and UvrCII, with distinct DNA binding activities. UvrCII, a tetramer, binds DNA but is inactive in incision, suggesting a regulatory role in nucleotide excision repair.
Area of Science:
- Molecular Biology
- Biochemistry
- DNA Repair
Background:
- The UvrC protein is essential for nucleotide excision repair (NER) in bacteria.
- Understanding the different forms and functions of UvrC is crucial for elucidating NER mechanisms.
Purpose of the Study:
- To characterize the distinct forms of UvrC protein purified from overproducing cells.
- To investigate the functional differences between UvrCI and UvrCII in DNA incision and binding.
Main Methods:
- Phosphocellulose and single-stranded DNA-cellulose chromatography for protein purification.
- Size exclusion chromatography and glutaraldehyde cross-linking to determine UvrCII's molecular weight.
- Nitrocellulose filter binding and gel retardation assays to assess DNA binding affinity.
Main Results:
- Two active forms of UvrC, UvrCI and UvrCII, were purified, with UvrCII being a tetramer of UvrCI.
- UvrCI and UvrCII incise UV-irradiated DNA similarly, but UvrCII's specific activity is lower.
- UvrCII binds to both damaged and undamaged double-stranded DNA, while UvrCI does not.
- UvrCII's DNA binding can inhibit its incision activity, which is restored by competitor DNA.
Conclusions:
- UvrC exists in at least two functional forms, UvrCI and UvrCII, with differential DNA binding properties.
- UvrCII's DNA binding may play a regulatory role in NER, potentially by influencing UvrA/UvrB complex formation.
- UvrCII might concentrate at genomic areas via double-stranded DNA binding, facilitating localized nucleotide excision repair.