Abstract
A 12-kDa and two 25-kDa polypeptides were isolated with highly purified calf thymus DNA polymerase delta by conventional chromatography. A 16-mer peptide sequence was obtained from the 12-kDa polypeptide which matched a new open reading frame from a human EST () encoding a hypothetical protein of unknown function. The protein was designated as p12. Human EST was identified as the putative human homologue of Schizosaccharomyces pombe Cdm1 by a tBlastn search of the EST data base using S. pombe Cdm1. The open reading frame of human EST encoded a polypeptide of 107 amino acids with a predicted molecular mass of 12.4 kDa, consistent with the experimental findings. p12 is 25% identical to S pombe Cdm1. Both of the 25-kDa polypeptide sequences matched the hypothetical KIAA0039 protein sequence, recently identified as the third subunit of pol delta. Western blotting of immunoaffinity purified calf thymus pol delta revealed the presence of p125, p50, p68 (the KIAA0039 product), and p12. With the identification of p12 mammalian pol delta can now be shown to consist of four subunits. These studies pave the way for more detailed analysis of the possible functions of the mammalian subunits of pol delta.
Highlights
DNA polymerase ␦ is the key polymerase that is involved in the replication of chromosomal DNA in eukaryotic cells
We have found that recombinant p125 catalytic subunit alone can only be stimulated by Proliferating cell nuclear antigen (PCNA) by 2-fold at most, while the overexpressed p125/p50 heterodimer is stimulated much less than pol ␦ purified by immunoaffinity chromatography [15, 16]
By peptide sequencing of polypeptides associated with the core pol ␦ in highly purified preparations isolated by p125 immunoaffinity chromatography, we have previously identified a 68-kDa polypeptide that is encoded by KIAA0039 and which is associated with the pol ␦ core
Summary
Materials—cDNA AA402118 was obtained from ATCC (Rockville, MD). Calf thymus tissue was obtained from Animal Technologies (Tyler, TX). Q-Sepharose, SP-Sepharose, heparin-Sepharose, Mono Q columns, and Mono S columns were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Purification of Calf Thymus Pol ␦—The immunoaffinity purification was performed as described previously by Jiang et al [18]. Conventional Purification of Calf Thymus Pol ␦—The following buffers were used: lysis buffer consisted of 50 mM Tris-HCl, pH 7.8, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.25 M sucrose, 5% glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml bacitracin, 10 mM benzamidine. TGEED buffer consisted of 50 mM Tris-HCl, pH 7.8, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and. DE 52 Q-Sepharose SP-Sepharose Mono Q Heparin-Sepharose Mono S Source Q15 Superdex 200
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