of Work: The nuclear genome is under constant attack from DNA damaging agents. Damaging agents such as cisplatinin and nitrogen mustard cause interstrand crosslinks in DNA that presents a difficult problem for replication and must be repaired for faithful replication and viability of the cell. A necessary step in repair of DNA crosslinks is the resynthesis after removal of the damaged DNA. It has been shown that most polymerases specialize in either replication of the genome or repair synthesis. We previously cloned the cDNA for the 8th human DNA polymerase, DNA polymerase q. The human cDNA encodes a putative DNA polymerase with a calculated molecular weight of 198 kDa. The derived protein sequence is homologous to the Drosophila melanogaster mus308 protein product, a DNA polymerase-helicase involved in repair of interstrand crosslinks. The full-length cDNA has been overexpressed in insect cells by a recombinant baculovirus and the protein partially purified as a histidine-tagged protein. The polymerase has maximum activity on polydA/oligodT, is dideoxy sensitive, NEM-resistant, and sensitive to salt. Amino acid sequence alignments predict several functions in this polypeptide, namely DNA polymerase, ATP binding and/or hydrolysis, and exonuclease function. Additionally, a single Tyr in motif B of the polymerase region predicts dideoxysensitivity and experimental data support this. To verify these activities we have changed essential amino acids in each one of these activity regions. The homologous metal-binding aspartic acids in exonuclease motif I have been changed to alanine to eliminate exonuclease activity. The essential metal binding aspartic acids in the DNA polymerase motif C have been changed to Ala to inactivate the polymerase function. The conserved Tyr in DNA polymerase motif B has been changed to Phe, presumable making the enzyme dideoxy-resistant. Finally, a critical Gly in Walker motif A has been changed to Asp to affect ATPase activity. The mutant proteins will be enzymatically characterized and compared to the wild type polymerase.
| 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 |
