A 5' to 3' exonuclease functionally interacts with calf DNA polymerase epsilon.

Abstract

Analysis of fractions containing purified DNA polymerase e from calf thymus has revealed the presence of a 5' to 3' exonuclease activity that is specific for a single strand of duplex DNA. This activity is capable of degrading a 3'-labeled oligonucleotide hybridized to M13mpl8 DNA. When a second oligonucleotide primer is anneal 3 bases upstream, degradation of the downstream primer is strictly dependent on DNA synthesis from the upstream primer. Replacement We have recently demonstrated the presence of two forms of DNA polymerase E in calf thymus extracts, designated E and E* (1). Both forms possess intrinsic DNA polymerase and single strand-specific 3' to 5' exonuclease activities. DNA polymerases E and 8* have molecular masses of 210 and 145 kDa, respectively. DNA polymerase E* can be proteolytically derived from DNA polymerase 8 during purification. We considered that this situation might be analogous to DNA polymerase I and the Klenow fragment from Escherichia coli (2, 3). In this case, limited proteolysis of DNA polymerase I results in removal of a domain containing the 5' to 3' exonuclease. The remaining DNA polymerase retains the capacity to exonucleolytically degrade single-stranded DNA in the 3' to 5' direction. However, we (4) and others (5) have examined the single-stranded exonuclease activity of DNA polymerase E from different sources and found it to demonstrate exclusively 3' to 5' directionality.Recent work on the 5' to 3' exonuclease of the DNA polymerase from Thermus aquaticus indicated that this activity is specific for one strand of double-stranded DNA (6, 7). Utilizing a template similar to that described by Longley et al. (6), we have detected a 5' to 3' exonucleolytic activity in fractions of DNA polymerase E. This activity has been purified to near homogeneity and its molecular mass has been determined to be 56 kDa as judged by SDS/PAGE. Thus, it appears that this activity represents a polypeptide other than polymerase E. Based on enzymatic and physical properties of the exonuclease, it may be a homolog of similar enzymes found in mouse cells (8) and HeLa cells (9). If so, in addition to the enzymologic similarity with the proteins described in these two reports, we find that the exonuclease specifically interacts with homologous DNA polymerase E, and not polymerases a and 8, in a two-primer assay described here. Based on these data, we propose a role for the complex of exonuclease and polymerase in DNA metabolism. MATERIALS AND METHODSMaterials. Synthetic oligodeoxyribonucleotides were from Genosys (The Woodlands, TX) and were purified by PAGE. The synthetic oligoribonucleotide corresponding to the -20 universal sequencing primer (5'-GUAAAACGACGGC-CAGU-3') was from National Bioscience Incorporated (Minneapolis). All nucleotides were Ultrapure from Pharmacia. The -20 universal sequencing primer (20 pmol) was 5' phosphorylated with T4 polynucleotide kinase (United States Biochemical) and 1 mM ATP in the buffer supplied by the manufacturer. The exonuclease substrate was created by...

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