Expression and Characterization of the Small Subunit of Human DNA Polymerase δ

A 12-kDa and two 25-kDa polypeptides were isolated with highly purified calf thymus DNA polymerase ␦ 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 (AA402118) encoding a hypothetical protein of unknown function. The protein was designated as p12. Human EST AA402118 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 AA402118 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 ␦. Western blotting of immunoaffinity purified calf thymus pol ␦ revealed the presence of p125, p50, p68 (the KIAA0039 product), and p12. With the identification of p12 mammalian pol ␦ 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 ␦.DNA polymerase ␦ (pol ␦) is the key polymerase that is involved in the replication of chromosomal DNA in eukaryotic cells. Studies of the in vitro replication of SV40 DNA have established that pol ␦ plays a central role in mammalian DNA replication (1). Proliferating cell nuclear antigen (PCNA), 1 the molecular sliding clamp of pol ␦, is a processivity factor for pol ␦ and ⑀ (2). PCNA was first identified as an activating factor for pol ␦ (3, 4) and is essential for replicative DNA synthesis. Several other factors have been identified as being required for SV40 DNA replication. Replication factor C (also known as activator-1) binds to the primer-template terminus, following which it recruits PCNA and then pol ␦ (5, 6) onto the DNA template. Replication Protein A, the single stranded DNAbinding protein, is involved in both initiation and elongation and also stimulates pol ␦ activity when replication factor C and PCNA are present (7,8). The current view of DNA replication at the replication fork is that the pol ␦ complex is responsible for synthesis of the leading strand and that pol ␦ also participates in synthesis of the lagging strand (1). DNA polymerase ␣/primase is primarily involved in the synthesis of RNA primers plus short stretches of DNA primers on the lagging strand, and the actual elongation of the primers is performed by DNA polymerase ␦ in a process that requires "polymerase switching" (9). Additional proteins, including topoisomerase and helicase activities, are also involved in the movement of the replication fork (1).Mammalian pol ␦ has been rigorously isolated by conventional methods as a heterodimer consisting of two subunits, p125 and p50 (3). The subunit structure of pol ␦ has been the focus of recent investigations in yeast, and these have led to the identification of additional subunits. In Schizosa...

mentioning

confidence: 45%

“…prokaryotic or lower eukaryotes such as yeast. Recently, expression systems for the p125 (15,24,25), the p50 subunit (16), and the recombinant heterodimer have been developed (26). The p50 subunit has no known enzymatic functions, but has been shown to be required for the response of the p125 subunit to PCNA (16,26).…”

Section: Discussionmentioning

confidence: 99%

Identification of a Fourth Subunit of Mammalian DNA Polymerase δ

Liu¹,

Mo²,

Rodriguez-Belmonte

et al. 2000

“…The enzyme we used in this study was purified by the immunoaffinity chromatography and gel filtration, and may contain other components besides the two subunit form that is isolated after stringent conventional chromatography (19). 4 In our hands, the pol ␦ p125 subunit overexpressed in Sf9 cells could be stimulated only 5-fold with PCNA in the presence of recombinant p50 expressed in E. coli (40). Polymerase ␦ obtained by co-expression of the p125 and p50 subunits (pol ␦ core) in the baculovirus system could be stimulated by PCNA at most 8-fold, far less than the stimulation obtained by the holoenzyme prepared from the immunoaffinity column.…”

Section: Table I Summary Of Mutations In the Interdomain Connector Lomentioning

confidence: 92%

The Interdomain Connector Loop of Human PCNA Is Involved in a Direct Interaction with Human Polymerase δ

Zhang¹,

Sun²,

Hsu

et al. 1998

Self Cite

Proliferating cell nuclear antigen (PCNA) is required for processive DNA synthesis catalyzed by DNA polymerase ␦ (pol ␦) and polymerase ⑀. We have shown that the epitope of a human PCNA inhibitory monoclonal antibody (74B1), which inhibits the PCNA stimulation of DNA synthesis catalyzed by pol ␦, maps to residues 121-135, which overlap the interdomain connector loop of PCNA (residues 119 -133). We have mutagenized residues 122-133 of human PCNA. The mutant proteins were expressed in Escherichia coli and purified to near-homogeneity. The interactions of the mutants with antibody 74B1 were examined; mutation of Gly-127 abolished the recognition by antibody 74B1 in a Western blot analysis, confirming the epitope assignment of 74B1. Mutations of Val-123, Leu-126, Gly-127, and Ile-128 affected the ability of PCNA to stimulate DNA synthesis by pol ␦ in several different assays. These mutations affected the interactions between PCNA and pol ␦ as determined by enzyme-linked immunosorbent assays. These mutants were also affected in their abilities to form a ternary complex with a DNA template-primer, as determined by electrophoretic mobility gel shift assays. The findings show that the interdomain connector loop region is involved in binding of pol ␦. This same region is involved in the binding of p21, and our findings support the view that the mechanism of inhibition of DNA synthesis by p21 is due to a competition for PCNA binding to pol ␦. DNA polymerase ␦ (pol ␦)1 is a central enzyme involved in the replication of mammalian chromosomal DNA and is also involved in DNA repair (1, 2). Studies of in vitro SV40 replication has established the role of pol ␦, as well as an understanding of the complex number of proteins that are involved in eukaryotic DNA replication (2). It is now considered that pol ␣ is involved in the lagging-strand synthesis, while pol ␦ is involved in leading-strand synthesis and also for completion of lagging strand synthesis (2). A key element of the function of a replicative DNA polymerase is the ability for extended processive synthesis of DNA. This ability is conferred on replicative polymerases by accessory proteins, which function as molecular sliding clamps. These clamps form toroidal rings that encircle the DNA strand and also interact with the polymerase. This function in Escherichia coli is fulfilled by the ␤ subunit (3), and in mammalian cells by PCNA (proliferating cell nuclear antigen), which is an essential factor for the eukaryotic DNA replication and functions as a processivity factor for pol ␦ (4, 5). The basis for the functions of the sliding clamps of both E. coli and PCNA has been elucidated by the determination of their crystal structures, which reveals them to be toroidal proteins that encircle the DNA strand (6, 7). PCNA is a homotrimer. Each subunit consists of two structurally equivalent domains giving the trimer a six-fold symmetry. PCNA is loaded onto double-stranded DNA by the action of RF-C (8), followed by the loading of pol ␦ through an interaction between PCNA and pol ␦...

“…Functional studies with recombinant p125 from human (28,39,40), mouse (41), and S. pombe (42) sources have shown that DNA synthesis catalyzed by p125 alone is not significantly stimulated by PCNA. On the other hand, PCNA was shown to stimulate the activity and processivity of the recombinant human heterodimer to the same extent as the native two-subunit enzyme isolated from calf thymus (28), suggesting the possibility that the interaction of PCNA with pol ␦ is mediated through the small subunit.…”

mentioning

confidence: 99%

Direct Interaction of Proliferating Cell Nuclear Antigen with the Small Subunit of DNA Polymerase δ

Lu¹,

Tan²,

Zhou³

et al. 2002

The interaction between proliferating cell nuclear antigen (PCNA) and DNA polymerase ␦ is essential for processive DNA synthesis during DNA replication/repair; however, the identity of the subunit of DNA polymerase ␦ that directly interacts with PCNA has not been resolved until now. In the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core DNA polymerase ␦, the 125-kDa catalytic subunit or the 50-kDa small subunit, directly interacts with PCNA. We found that PCNA co-immunoprecipitated with human p50, as well as calf thymus DNA polymerase ␦ heterodimer, but not with p125 alone, suggesting that PCNA directly interacts with p50 but not with p125. A PCNA-binding motif, similar to the sliding clamp-binding motif of bacteriophage RB69 DNA polymerase, was identified in the N terminus of p50. A 22-amino acid oligopeptide containing this sequence (MRPFL) was shown to bind PCNA by far Western analysis and to compete with p50 for binding to PCNA in co-immunoprecipitation experiments. The binding of p50 to PCNA was inhibited by p21, suggesting that the two proteins compete for the same binding site on PCNA. These results establish that the interaction of PCNA with DNA polymerase ␦ is mediated through the small subunit of the enzyme.