The yeast two-hybrid screening method was used to identify novel proteins that associate with human DNA polymerase ␦ (pol ␦). Two baits were used in this study. These were the large (p125) and small (p50) subunits of the core pol ␦ heterodimer. p50 was the only positive isolated with p125 as the bait. Two novel protein partners, named PDIP38 and PDIP46, were identified from the p50 screen. In this study, the interaction of PDIP38 with pol ␦ was further characterized. PDIP38 encodes a protein of 368 amino acids whose C terminus is conserved with the bacterial APAG protein and with the F box A protein. It was found that PDIP38 also interacts with proliferating cell nuclear antigen (PCNA). The ability of PDIP38 to interact with both the p50 subunit of pol ␦ and with PCNA was confirmed by pull-down assays using glutathione S-transferase (GST)-PDIP38 fusion proteins. The PCNA-PDIP38 interaction was also demonstrated by PCNA overlay experiments. The association of PDIP38 with pol ␦ was shown to occur in calf thymus tissue and mammalian cell extracts by GST-PDIP38 pull-down and coimmunoprecipitation experiments. PDIP38 was associated with pol ␦ isolated by immunoaffinity chromatography. The association of PDIP38 with pol ␦ could also be demonstrated by native gel electrophoresis.DNA replication is essential not only for duplication of the genome but also for maintenance of genomic integrity during DNA repair (1, 2). Chromosomal DNA replication in eukaryotic cells requires three distinct DNA polymerases-␣, -␦, and -⑀ (1-6). pol 1 ␦ is required for replication of the leading strand and for completion of the lagging strand synthesis at the replication fork (7). The action of pol ␦ as a processive enzyme requires its interaction with proliferating cell nuclear antigen (PCNA), which functions as a molecular sliding clamp (1). The core mammalian pol ␦ enzyme consists of a tightly associated heterodimer of 125-and 50-kDa subunits. pol ␦ has been shown recently (8 -14) to consist of at least four subunits, consisting of the core enzyme and two additional subunits in both yeast and mammalian systems. In the yeast Schizosaccharomyces pombe, Cdc27 and Cdm1 have been identified as the third and the fourth pol ␦ subunits, respectively, (8, 9). In Saccharomyces cerevisiae Pol32p has been identified as the homologue of the S. pombe third subunit (10). A human homologue of Cdc27, the KIAA0039 gene product (11-13), and p12, a human homologue of Cdm1 (14), have recently been identified and can be considered to be the third and fourth subunits of human pol ␦. Our laboratory has been interested in the identification of additional protein components that are involved in the formation of the pol ␦ replication complex. In this study we report the identification of two novel proteins, PDIP38 and PDIP46, that interact with the p50 subunit of pol ␦ by the use of the yeast two-hybrid (15, 16) screening method. PDIP38 was shown to be a PCNA-binding protein, and its interaction with pol ␦ was established by additional experiments. EXPERIMENTAL PR...

The formation of a complex between DNA polymerase ␦ (pol ␦) and its sliding clamp, proliferating cell nuclear antigen (PCNA), is responsible for the maintenance of processive DNA synthesis at the leading strand of the replication fork. In this study, the ability of the p125 catalytic subunit of DNA polymerase ␦ to engage in protein-protein interactions with PCNA was established by biochemical and genetic methods. p125 and PCNA were shown to co-immunoprecipitate from either calf thymus or HeLa extracts, or when they were ectopically co-expressed in Cos 7 cells. Because pol ␦ is a multimeric protein, this interaction could be indirect. Thus, rigorous evidence was sought for a direct interaction of the p125 catalytic subunit and PCNA. To do this, the ability of recombinant p125 to interact with PCNA was established by biochemical means. p125 co-expressed with PCNA in Sf9 cells was shown to form a physical complex that can be detected on gel filtration and that can be cross-linked with the bifunctional cross-linking agent Sulfo-EGS (ethylene glycol bis (sulfosuccinimidylsuccinate)). An interaction between p125 and PCNA could also be demonstrated in the yeast two hybrid system. Overlay experiments using biotinylated PCNA showed that the free p125 subunit interacts with PCNA. The PCNA overlay blotting method was also used to demonstrate the binding of synthetic peptides corresponding to the N2 region of pol ␦ and provides evidence for a site on pol ␦ that is involved in the protein-protein interactions between PCNA and pol ␦. This region contains a sequence that is a potential member of the PCNA binding motif found in other PCNA-binding proteins. These studies provide an unequivocal demonstration that the p125 subunit of pol ␦ interacts with PCNA.Proliferating cell nuclear antigen (PCNA) 1 was originally discovered as an antigen in autoimmune sera from patients with systemic lupus erythematosus and was reported to be found only in actively proliferating cells (1). It was later shown to be a factor that enhanced the processivity of DNA polymerase ␦ (pol ␦) and to have key roles in both DNA replication and repair (2, 3). There have been striking recent advances in our understanding of the structure and functions of PCNA (4). Purification and expression of human recombinant PCNA and its physiochemical characterization established that it was a trimeric protein (5). The crystal structures of both yeast and human PCNA have been determined (6, 7). Like the T4 gene 45 protein and the ␤ subunit of Escherichia coli DNA polymerase III holoenzyme, PCNA functions as a sliding DNA clamp that forms a closed ring around duplex DNA (8). The binding of pol ␦ to PCNA provides an elegant micromechanical solution to the biological need to maintain an extraordinarily high level of processivity during the synthesis of chromosomal DNA (8 -10). Recently, it has also been found that PCNA has a number of protein partners with which it interacts (4, 9, 11). Pol ␦ has been shown to be involved not only in DNA replication but also in DNA repair a...

Mammalian DNA polymerase delta was originally characterized as a tightly associated heterodimer consisting of the catalytic subunit, p125, and the p50 subunit. Recently, two additional subunits, the third (p68) and fourth subunits (p12), have been identified. The heterotetrameric human pol delta complex was reconstituted by overexpression of the four subunits in Sf9 cells, followed by purification to near-homogeneity using FPLC chromatography. The properties of the four-subunit enzyme were shown to be functionally indistinguishable from those of pol delta isolated from calf thymus. The physicochemical properties of both the reconstituted heterotetramer and the heterodimer of the p125 and p50 subunits were examined by gel filtration and glycerol gradient ultracentrifugation. These studies show quite clearly that the heterodimer and heterotetramer complexes do not behave in solution as dimeric structures. This issue is of significance because several studies of the yeast pol delta complexes have indicated that the third subunit is able to bring about the dimerization of the pol delta complex. The heterodimer is only weakly stimulated by PCNA, whereas the heterotetramer is strongly stimulated to a level with a specific activity comparable to that of the calf thymus enzyme. These results resolve earlier, conflicting reports on the response of the heterodimer to PCNA. Nevertheless, the heterodimer does have some ability to interact functionally with PCNA, consistent with evidence that the p125 subunit itself has an ability to interact with PCNA. The functional interaction of PCNA with the pol delta complex may likely involve multiple contacts.

The actin filament-associated protein and Src-binding partner, AFAP-110, is an adaptor protein that links signaling molecules to actin filaments. AFAP-110 binds actin filaments directly and multimerizes through a leucine zipper motif. Cellular signals downstream of Src 527F can regulate multimerization. Here, we determined recombinant AFAP-110 (rAFAP-110)-bound actin filaments cooperatively, through a lateral association. We demonstrate rAFAP-110 has the capability to cross-link actin filaments, and this ability is dependent on the integrity of the carboxy terminal actin binding domain. Deletion of the leucine zipper motif or PKC phosphorylation affected AFAP-110's conformation, which correlated with changes in multimerization and increased the capability of rAFAP-110 to cross-link actin filaments. AFAP-110 is both a substrate and binding partner of PKC. On PKC activation, stress filament organization is lost, motility structures form, and AFAP-110 colocalizes strongly with motility structures. Expression of a deletion mutant of AFAP-110 that is unable to bind PKC blocked the effect of PMA on actin filaments. We hypothesize that upon PKC activation, AFAP-110 can be cooperatively recruited to newly forming actin filaments, like those that exist in cell motility structures, and that PKC phosphorylation effects a conformational change that may enable AFAP-110 to promote actin filament cross-linking at the cell membrane.

DNA polymerase delta, the key enzyme for eukaryotic chromosomal replication, has been well characterized as consisting of a core enzyme of a 125 kDa catalytic subunit and a smaller 50 kDa subunit. However, less is known about the other proteins that may comprise additional subunits or participate in the macromolecular protein complex that is involved in chromosomal DNA replication. In this study, the properties of calf thymus pol delta preparations isolated by immunoaffinity chromatography were investigated. It is demonstrated for the first time using highly purified preparations that the pol delta heterodimer is associated with other polypeptides in high-molecular weight species that range from 260000 to >500000 in size, as determined by FPLC gel filtration. These preparations are associated with polypeptides of ca. 68-70, 34, 32, and 25 kDa. Similar findings were revealed with glycerol gradient ultracentrifugation. The p68 polypeptide was shown to be a PCNA binding protein by overlay methods with biotinylated PCNA. Protein sequencing of the p68, p34, and p25 polypeptide bands revealed sequences that correspond to the hypothetical protein KIAA0039. KIAA0039 displays a small but significant degree of homology to Schizosaccharomyces pombe Cdc27, which, like Saccharomyces cerevisiae Pol32p, has been described as the third subunit of yeast pol delta. These studies provide evidence that p68 is a subunit of pol delta. In addition, the p68-70 and p32 polypeptides were found to be derived from the 70 and 32 kDa subunits of RPA, respectively.