of Work: Exogenous DNA damaging agents such as cisplatinin, nitrogen mustard, and psoralen create interstrand DNA crosslinks. Such non-coding lesions must be repaired to ensure accurate replication of the genome and viability of the organism. The Drosophila mus308 mutation, which confers hypersensitivity to nitrogen mustard but not the monofunctional agent methyl-methane sulfonate, identified a DNA polymerase likely involved in processing DNA crosslinks. Based on homology to the Drosophila mus308 gene and another Family A DNA polymerase, DNA polymerase g, we previously cloned and expressed the cDNA for human DNA polymerase q. Recent analysis of the gene for human DNA polymerase theta has identified a 9 kb coding region, encoding the full length DNA polymerase theta with a molecular weight of 300 kDa. Amino acid sequence alignments predict DNA polymerase, ATP binding and/or hydrolysis, helicase activity, and 3' to 5' exonuclease functions for this enzyme. We previously have cloned and expressed cDNA constructs of 6.5 and 3 kb, which have produced functional DNA polymerase polypeptides of 200 and 100 kDa. These have been produced in baculovirus and in E. coli. The substrate specificity and accuracy of DNA synthesis in vitro were determined. Unlike many of the other newly discovered DNA polymerases, we determined that polymerase theta synthesizes DNA with high fidelity. Site directed alteration of conserved amino acids have identified the polymerase activity in these polypeptides. Our current effort is to isolate a full-length cDNA encoding the full-length 300 kDa polypeptide. We recently isolated another 1.8 kbp cDNA clone, giving the total cDNA isolated of 7.8 kbp.
| Burgers, P M; Koonin, E V; Bruford, E et al. (2001) Eukaryotic DNA polymerases: proposal for a revised nomenclature. J Biol Chem 276:43487-90 |
