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Identification of a mammalian nuclear factor and human cDNA-encoded proteins that recognize DNA containing apurinic
J Lenz1, S A Okenquist, J E LoSardo
1Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461.
Abstract:
Damage to DNA can have lethal or mutagenic consequences for cells unless it is detected and repaired by cellular proteins. Repair depends on the ability of cellular factors to distinguish the damaged sites. Electrophoretic binding assays were used to identify a factor from the nuclei of mammalian cells that bound to DNA containing apurinic sites. A binding assay based on the use of beta-galactosidase fusion proteins was subsequently used to isolate recombinant clones of human cDNAs that encoded apurinic DNA-binding proteins. Two distinct human cDNAs were identified that encoded proteins that bound apurinic DNA preferentially over undamaged, methylated, or UV-irradiated DNA. These approaches may offer a general method for the detection of proteins that recognize various types of DNA damage and for the cloning of genes encoding such proteins.
Insights
Researchers identified human proteins that specifically bind to damaged DNA, particularly apurinic sites. This discovery aids in understanding DNA repair mechanisms and cloning relevant genes.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA damage can cause cell death or mutations if not repaired.
- Cellular proteins must distinguish damaged DNA sites for effective repair.
Purpose of the Study:
- To identify mammalian nuclear factors that bind to DNA with apurinic sites.
- To clone human complementary DNAs (cDNAs) encoding apurinic DNA-binding proteins.
Main Methods:
- Electrophoretic mobility shift assays (EMSA) were used to detect DNA-binding factors.
- Beta-galactosidase fusion protein assays facilitated the isolation of human cDNA clones.
Main Results:
- A nuclear factor binding to apurinic DNA sites was identified.
- Two distinct human cDNAs encoding proteins with preferential binding to apurinic DNA were isolated.
- These proteins showed higher affinity for apurinic DNA compared to undamaged, methylated, or UV-irradiated DNA.
Conclusions:
- The study successfully identified and cloned human proteins that recognize apurinic DNA sites.
- The methods developed may serve as a general approach for detecting DNA damage-recognizing proteins and cloning their genes.