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Studies of the murine DDB1 and DDB2 genes
1Division of Biochemistry, Barker Hall, University of California, Berkeley, CA, USA.
Abstract:
Human DDB (Damaged DNA Binding protein) is a heterodimer of 48 and 127kDa subunits whose activity is absent from cell strains derived from a subset of Xeroderma Pigmentosum (XP) complementation group E individuals (Ddb(-)) [Keeney, S., Wein, H., and Linn, S., (1992). Mut. Res. 273, 49-56]. Whereas in vivo DNA repair appears to be compromised in both Ddb(-) and Ddb(+) XPE cells, DDB activity is not necessary for nucleotide excision repair (NER) in vitro. In this study, the presence of a specific UV-damaged DNA binding activity in mouse cell-free extracts that is comparable to the activity observed in HeLa cells was demonstrated. The mouse DDB2 cDNA, coding for DDB p48 subunit, was cloned and the partial genomic structure of DDB2 was obtained. A search of current databases revealed amino acid sequences of mouse and Drosophila predicted p127 homologues, but not of a Drosophila p48 homologue. The alignment of these higher eukaryotic p127 sequences uncovered the presence of three highly conserved domains in the p127 polypeptides which we hypothesize could function in DNA binding, transcription-transactivation, and protein-protein interaction, respectively.
Insights
Researchers identified UV-damaged DNA binding protein (DDB) activity in mouse cells, cloning the mouse DDB2 gene. They also found conserved domains in p127 homologues, suggesting roles in DNA binding and transcription.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Human Damaged DNA Binding protein (DDB) is a heterodimer crucial for DNA repair.
- Deficiency in DDB activity is linked to Xeroderma Pigmentosum (XP) group E.
- While DDB is vital in vivo, its necessity for in vitro nucleotide excision repair (NER) is unclear.
Purpose of the Study:
- To investigate UV-damaged DNA binding activity in mouse cell-free extracts.
- To clone the mouse DDB2 cDNA, encoding the DDB p48 subunit.
- To analyze conserved domains in p127 homologues across higher eukaryotes.
Main Methods:
- Demonstration of specific UV-damaged DNA binding activity in mouse extracts.
- Cloning of mouse DDB2 cDNA and determination of its partial genomic structure.
- Bioinformatic analysis of p127 homologue sequences from mouse and Drosophila.
Main Results:
- Mouse cell-free extracts exhibit UV-damaged DNA binding activity similar to HeLa cells.
- The mouse DDB2 cDNA, encoding the DDB p48 subunit, was successfully cloned.
- Three conserved domains were identified in higher eukaryotic p127 homologues, potentially involved in DNA binding, transcription, and protein interactions.
Conclusions:
- Mouse cells possess functional DDB activity comparable to human cells.
- The conserved domains in p127 homologues suggest fundamental roles in DNA processing and gene regulation.
- Further research is warranted to elucidate the precise functions of these conserved domains.