Abstract
The cDNA encoding the catalytic polypeptide of human DNA polymerase epsilon was cloned. The deduced amino acid sequence reveals that the catalytic polypeptide is 2257 amino acids in length and its calculated molecular mass is 258 kDa. A single RNA message of 7.5 kilobases was recognized by isolated cDNA clones. The identity of the cDNA was verified by direct amino acid sequencing of tryptic fragments derived from the catalytic polypeptide of the HeLa DNA polymerase epsilon. The primary structure comparison with multiple DNA polymerases indicates that human DNA polymerase epsilon catalytic polypeptide is a homolog of the yeast Saccharomyces cerevisiae DNA polymerase II catalytic polypeptide. The proteins are 39% identical. In the region containing known DNA polymerase consensus motifs, the identity is 63%. The expression of the mRNA encoding DNA polymerase epsilon is strongly dependent on cell proliferation.
Highlights
The cDNAencoding the catalyticpolypeptide of human DNApolymerase t was cloned
CIA(AG)ICC(CT)TTIA(AG)(CT)TCwith a Sac1 restriction site and three extra nucleotides in the 5' terminus was prepared according to the amino acid sequenEceLKGFE of the yeast DNA polymerases I1 (aminoacidresidues 980-985 (Morrison et al, 1990)) by replacing some of the degenerate nucleotideswithdeoxyinosine (I) (Takahashi et al, 1985)
Due to a minor heterogeneity of the sequenced polypeptides, strong signals from 2 amino acids were obtained at some sequencing cycles
Summary
Ture, loaded in asingle8-cm-wide well on a 1.5-mm 6 . 5 7 SDSpolyacrylamide gel, and electrophoresed with a 40-mA current essen-. Strain and Cell Lines-Escherichia coli strains XL1-Blue (recAl tially as described (Laemmli, 1970). TnlO(tetR)I)and Y1089 (A(lac)U169 A(lon) araD139strA hfl- pm nitrocellulose membrane as described Plasmid P2A1,which contains a fragment of localized by staining with Ponceau S, the strip containing protein genomic DNA encoding S. cereuisiae DNA polymerase I1 Nitrocellulose pieceswere removed, and trifluoroacetic acid was added to a concentration of 1%to the solution containing tryptic peptides. The peptides were eluted a t a flow rate of 150pllmin with a linear acetonitrile gradient from 0 to 60% supplemented with 0.1% trifluoroacetic acid. Amino Acid Sequence Alignments-Amino acid sequence alignments were performed with the GeneWorks program (IntelliGenetics Inc., Mountain View, CA).DNA polymerase sequences used for alignment were yeast pol I1 (Morrison et al, 1990), human pol CY Amino Acid Sequence Alignments-Amino acid sequence alignments were performed with the GeneWorks program (IntelliGenetics Inc., Mountain View, CA).DNA polymerase sequences used for alignment were yeast pol I1 (Morrison et al, 1990), human pol CY (Wong et ~ l . 1, 9881, yeast pol I (Pizzagalli et al, 1988), human pol d (Yang et al, 1992), yeast pol 111 (Boulet et al, 1989; Morrison and Sugino, 1991),Epstein-Barr virus (Baer et QL, 1984),herpes simplex virus (Larder et al, 1987), adenovirus (Alestrom et al, 1982), baculovirus (Tomalski et d., 1988),bacteriophage T4 (Spicer et al.,1988), bacteriophage $29 (Yoshikawa and Ito, 1982), and bacteriophage PRDl (Savilahti and Bamford, 1987)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
