The identity of DNA replication proteins and cell cycle regulatory proteins which can be found in complexes involving PCNA were investigated by the use of PCNA immobilized on Sepharose 4B. A column containing bovine serum albumin (BSA) bound to Sepharose was used as a control. Fetal calf thymus extracts were chromatographed on PCNA-Sepharose and BSA-Sepharose. The columns were washed and then eluted with 0.5 M KCl. The salt eluates were examined for the presence of both DNA replication proteins (Pol alpha, delta, straightepsilon, PCNA, RFC, RFA, DNA ligase I, NDH II, Topo I and Topo II) and cell cycle proteins (Cyclins A, B1, D1, D2, D3, E, CDK2, CDK4, CDK5 and p21) by western blotting with specific antibodies. The DNA replication proteins which bound to PCNA-Sepharose included DNA polymerase delta and straightepsilon, PCNA, the 37 and 40 kDa subunits of RFC, the 70 kDa subunit of RPA, NDH II and topoisomerase I. No evidence for the binding of DNA polymerase alpha, DNA ligase I or topoisomerase II was obtained. Of the cell cycle proteins investigated, CDK2, CDK4 and CDK5 were bound. This study presents strong evidence that PCNA is a component of protein complexes containing DNA replication, repair and cell cycle regulatory proteins.

The catalytic subunit of human DNA polymerase (pol) ␦ was overexpressed in an active, soluble form by the use of a baculovirus system in insect cells. The recombinant enzyme was separated from endogenous DNA polymerases by phosphocellulose, Mono Q-Sepharose, and single-stranded DNA-cellulose chromatography. Recombinant DNA pol ␦ was also purified by immunoaffinity chromatography. The enzymatic properties of the purified catalytic subunit were characterized. The enzyme was active and possessed both DNA polymerase and associated 3 to 5 exonuclease activities. NH 2 -terminal deletion mutants retained polymerase activity, whereas the core and COOH-terminal deletion mutants were devoid of any measurable activities. Coinfection of Sf9 cells with recombinant baculovirus vectors for pol ␦ and cyclin-dependent kinase (cdk)-cyclins followed by metabolic labeling with 32 P i showed that the recombinant catalytic subunit of pol ␦ could be hyperphosphorylated by G 1 phase-specific cdk-cyclins. When cdk2 was coexpressed with pol ␦ in Sf9 cells, pol ␦ was found to coimmunoprecipitate with antibodies against cdk2. Experiments with deletion mutants of pol ␦ showed that the NH 2 -terminal region was essential for this interaction. Coimmunoprecipitation and Western blot experiments in Molt 4 cells confirmed the interaction in vivo. Preliminary experiments showed that phosphorylation of the catalytic subunit of pol ␦ by cdk2-cyclins had little or no effect on the specific activity of the enzyme. DNA polymerase (pol)1 ␦ is the central enzyme in eukaryotic DNA replication (1) and also serves an important role in DNA repair (2). Isolation of the calf thymus (3) and human (4) enzymes has shown that it consists of at least two core subunits of 125 and 50 kDa. The hallmarks of this polymerase are that it has an intrinsic 3Ј to 5Ј exonuclease activity, distinguishing it from pol ␣ and pol ␤. The 125-kDa subunit of human pol ␦ (p125) has been identified as the catalytic subunit (4). Pol ␦ is a member of a family of DNA polymerases which includes DNA polymerase ␣, pol ⑀, the herpesvirus DNA polymerases, and bacteriophage T4 polymerase (5, 6). Examination of the regions of conserved sequence has led to the identification of domains that are potentially required for DNA interaction, deoxynucleotide interaction, as well as the 3Ј to 5Ј exonuclease activity of pol ␦ (7). In addition, there are several regions in the NH 2 and COOH termini which are conserved among human pol ␦, yeast pol ␦, and yeast and human pol ⑀ (5, 7).Studies of the replication of SV40 DNA in vitro have led to the identification of a number of accessory proteins, which, together with pol ␦, are required for the formation of a replication complex at the replication fork. These include PCNA, which functions as a sliding clamp and enhances the processivity of pol ␦, consistent with its role as the leading strand polymerase (8). Although there have been some mutagenesis studies of the yeast pol ␦ (9), little has been done with human or mammalian pol ␦, largely because of the lac...

DNA polymerase ␦ is a heterodimer consisting of a catalytic subunit of 125 kDa and a small subunit of 50 kDa (p50). We have overexpressed p50 in Escherichia coli and have characterized the recombinant protein. p50 was readily overexpressed using the pET vector as an insoluble protein. A procedure was developed for its purification and renaturation. Examination of the physicochemical properties of renatured p50 showed that it is a monomeric protein with an apparent molecular weight of 60,000, a Stokes radius of 34 Å, and a sedimentation coefficient of 4.1 S. Its physical properties were indistinguishable from p50 expressed as a soluble protein using the pTACTAC vector. Examination of the effects of recombinant p50 on the activity of DNA polymerase ␦ showed that p50 is able to slightly stimulate (about 2-fold) the activity of the recombinant 125-kDa catalytic subunit using poly(dA)⅐oligo(dT) as a template in the absence of proliferating cell nuclear antigen. In the presence of proliferating cell nulear antigen, activity is stimulated about 5-fold. Seven stable hybridoma cell lines were established that produced monoclonal antibodies against p50. One of these antibodies (13D5) inhibited the activity of calf thymus DNA polymerase ␦. This antibody, when coupled to a solid support, also was found to provide a method for the immunoafffinity purification of recombinant p50 and of DNA polymerase ␦ from calf thymus or HeLa extracts. Immunoprecipitation and enzyme-linked immunosorbent assays also confirmed that p50 interacts with the catalytic subunit of DNA polymerase ␦.DNA polymerase (pol) 1 ␦ is involved in both DNA replication (1) and DNA repair (2) in mammalian cells. Its activity is stimulated by PCNA, a DNA sliding clamp which provides the processivity required for its role in DNA replication (3, 4). In addition to PCNA, a number of other accessory proteins are required for the assembly of a functional replication complex at the leading strand of the replication fork. These include the multi-subunit replication factor C (RF-C) complex and replication protein A (RP-A), a single-stranded DNA-binding protein (5). pol ␦ isolated from calf thymus (6) and human placenta (7) is a heterodimer of 125-and 50-kDa polypeptides. The catalytic activity had been clearly demonstrated to be associated with the 125-kDa subunit (7), and preparations of mammalian pol ␦ have been reported that contain only the active 125-kDa subunit (8, 9). Goulian et al. (9) isolated two preparations of mouse DNA polymerase ␦, one of which consisted of a single polypeptide of 123-125 kDa and was unresponsive to PCNA stimulation. It was suggested that the 50-kDa polypeptide is required for the stimulation of DNA polymerase ␦ by PCNA (9). However, yeast pol ␦ catalytic subunit overexpressed in Escherichia coli was stimulated 2.5-to 3-fold (10). Human pol ␦ catalytic subunit expressed in the vaccinia virus system also showed a slight increase in activity in the presence of PCNA (11). This increase in activity is significantly lower than the 34-fold stimul...

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