As first observed by Wittenberg (Kesti, T., Flick, K., Keranen, S., Syvaoja, J. E., and Wittenburg, C. (1999) Mol. Cell 3, 679 -685), we find that deletion mutants lacking the entire N-terminal DNA polymerase domain of yeast pol ⑀ are viable. However, we now show that point mutations in DNA polymerase catalytic residues of pol ⑀ are lethal. Taken together, the phenotypes of the deletion and the point mutants suggest that the polymerase of pol ⑀ may normally participate in DNA replication but that another polymerase can substitute in its complete absence. Substitution is inefficient because the deletion mutants have serious defects in DNA replication. This observation raises the question of what is the essential function of the C-terminal half of pol ⑀. We show that the ability of the C-terminal half of the polymerase to support growth is disrupted by mutations in the cysteinerich region, which disrupts both dimerization of the POL2 gene product and interaction with the essential DPB2 subunit, suggesting that this region plays an important architectural role at the replication fork even in the absence of the polymerase function. Finally, the S phase checkpoint, with respect to both induction of RNR3 transcription and cell cycle arrest, is intact in cells where replication is supported only by the C-terminal half of pol ⑀, but it is disrupted in mutants affecting the cysteine-rich region, suggesting that this domain directly affects the checkpoint rather than acting through the N-terminal polymerase active site.In Saccharomyces cerevisiae, three DNA polymerases participate in chromosomal DNA replication, pol 1 ␣, pol ␦, and pol ⑀. pol ␣ is primarily involved in the initiation of DNA replication and priming of Okazaki fragments (2), whereas pol ␦ and pol ⑀ are required for completion of synthesis of both the leading and lagging strands. The precise reactions performed by pol ␦ and pol ⑀ on leading and lagging strands, however, have not yet been delineated. In an interesting contrast to yeast chromosomes, simian virus 40 DNA replication does not require pol ⑀. Instead, pol ␣ and pol ␦ are sufficient for viral DNA replication (3). Thus, there appears to be some plasticity in the eukaryotic replication fork.Pol ⑀ is a multi-subunit complex consisting of Pol2p, Dpb2p, Dpb3p, and Dpb4p (4). The Pol2p is the catalytic subunit, and it is encoded by the POL2 gene (5). The Pol2p is a class B polymerase, characterized by a series of conserved domains, called domains I-VI, containing the exonuclease subdomains and the DNA polymerase active site residues in the N-terminal half of the protein (Fig. 1A) (6, 7). Mutations M643I and P710S (the pol2-9 and pol2-18 alleles, respectively) within the polymerase domain in POL2 result in temperature sensitivity (8). The remaining half of POL2 consists of a long region that is conserved in pol ⑀ from all organisms but is not found in any other class B polymerase. An interesting feature of the extreme C terminus is a cysteine-rich stretch of amino acids containing two putative zinc fingers...

It has been proposed that C-terminal motifs of the catalytic subunit of budding yeast polymerase (pol) ⑀ (POL2) couple DNA replication to the S/M checkpoint (Navas, T. A., Zheng, Z., and Elledge, S. J. (1995) Cell 80, 29 -39). Scanning deletion analysis of the C terminus reveals that 20 amino acid residues between two putative C-terminal zinc fingers are essential for DNA replication and for an intact S/M cell cycle checkpoint. All mutations affecting the inter-zinc finger amino acids or the zinc fingers themselves are sensitive to methylmethane sulfonate and have reduced ability to induce RNR3, showing that the mutants are defective in the transcriptional response to DNA damage as well as the cell cycle response. The mutations affect the assembly of the pol ⑀ holoenzyme. Two-hybrid assays show that the POL2 subunit interacts with itself, and that the replication and checkpoint mutants are specifically defective in the interaction, suggesting (but not proving) that direct or indirect dimerization may be important for the normal functions of pol ⑀. The POL2 C terminus is sufficient for interaction with DPB2, the essential and phylogenetically conserved subunit of pol ⑀, but not for interaction with DPB3. Neither Dpb3p nor Dpb2p homodimerizes in the two-hybrid assay.

Human immunodeficiency virus (HIV-1) is able to recombine by transfer of the growing DNA strand from internal regions of one genome to another. The strand transfer reaction, catalyzed by HIV-1 reverse transcriptase (RT), was conducted in vitro between donor and acceptor RNA templates that were derived from natural HIV-1 nef genes. The donor and acceptor templates shared a nearly homologous region where strand transfer could occur, differing only in that the acceptor had a 36-nucleotide insertion and 6 widely spaced base substitutions compared with the donor. We sequenced elongated primers that underwent transfer. The position of transfer was revealed by the change of sequence from that of the donor to that of the acceptor. Results showed a positive correlation between positions where the RT paused during synthesis and enhancement of strand transfer. Elimination of a pause site, with a minimal change in sequence, decreased the frequency of strand transfer in the immediate area. Analysis of the sequence of DNA products resulting from transfer at a frequently used site showed that mutations had been introduced into the DNA at about the point of transfer. Remarkably, approximately 30% of the products contained mutations. Base substitutions, short additions and deletions were observed. Mutations did not appear in DNA products extended on the donor template without transfer. The identity of the mutations suggests that they were caused by a combination of slippage and non-template-directed nucleotide addition. These results indicated that the detected mutations were related to the process of strand transfer.

Saccharomyces cerevisiae DNA polymerase epsilon (pol ⑀) is essential for chromosomal replication. A major form of pol ⑀ purified from yeast consists of at least four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. We have investigated the protein/protein interactions between these polypeptides by using expression of individual subunits in baculovirus-infected Sf9 insect cells and by using the yeast two-hybrid assay. The essential subunits, Pol2p and Dpb2p, interact directly in the absence of the other two subunits, and the C-terminal half of POL2, the only essential portion of Pol2p, is sufficient for interaction with Dpb2p. Dpb3p and Dpb4p, non-essential subunits, also interact directly with each other in the absence of the other two subunits. We propose that Pol2p⅐Dpb2p and Dpb3p⅐Dpb4p complexes interact with each other and document several interactions between individual members of the two respective complexes. We present biochemical evidence to support the proposal that pol ⑀ may be dimeric in vivo. Gel filtration of the Pol2p⅐Dpb2p complexes reveals a novel heterotetrameric form, consisting of two heterodimers of Pol2p⅐Dpb2p. Dpb2p, but not Pol2p, exists as a homodimer, and thus the Pol2p dimerization may be mediated by Dpb2p. The pol2-E and pol2-F mutations that cause replication defects in vivo weaken the interaction between Pol2p and Dpb2p and also reduce dimerization of Pol2p. This suggests, but does not prove, that dimerization may also occur in vivo and be essential for DNA replication.DNA polymerases (pol) 1 play essential roles in the duplication of genetic material and DNA repair in both prokaryotes and eukaryotes (1). In Saccharomyces cerevisiae there exist three essential nuclear DNA polymerases, pol ␣, ␦, and ⑀ (2-4). Despite years of study, several perhaps equally plausible models exist for the function of each polymerase during DNA replication. Pol ␣ plays the role of a primase in the initiation of DNA replication on both leading and lagging strands in the simian virus 40 in vitro system (5). Pol ␦ and pol ⑀ are required for the bulk of the replication on the leading and lagging strands (6 -8). The precise location of pol ␦ and pol ⑀ on leading and lagging strands, however, is not known. Pol ␣, pol ␦, and pol ⑀ have also been shown to participate in DNA repair (1).Polymerase ⑀ was the first proofreading polymerase purified from yeast (2). Since then it has been purified from a number of sources including Schizosaccharomyces pombe, the silkworm Bombyx mori, and HeLa cells (3-7). The S. cerevisiae pol ⑀ consists of four subunits, Pol2p, Dpb2p, Dpb3p, and Dpb4p, with an estimated stoichiometry of 1:1:4:4 (4). These four proteins are encoded by the POL2, DPB2, DPB3, and DPB4 genes (4, 8). 2 The POL2 gene encodes the 256-kDa catalytic subunit of pol ⑀ (8). Mammalian and S. pombe Pol2p show strong sequence similarity to yeast Pol2p (7, 9). The DPB2, DPB3, and DPB4 genes encode the remaining 80-, 34-, and 29-kDa subunits of the yeast pol ⑀ holoenzyme, respectively (4, 10, 11). 2 A functional and structural...

DNA polymerase ⑀ (pol ⑀) is a multiple subunit complex consisting of at least four proteins, including catalytic Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol ⑀ has been shown to play essential roles in chromosomal DNA replication. Here, we report reconstitution of the yeast pol ⑀ complex, which was expressed and purified from baculovirus-infected insect cells. During the purification, we were able to resolve the pol ⑀ complex and truncated Pol2p (140 kDa), as was observed initially with the pol ⑀ purified from yeast. Biochemical characterization of subunit stoichiometry, salt sensitivity, processivity, and stimulation by proliferating cell nuclear antigen indicates that the reconstituted pol ⑀ is functionally identical to native pol ⑀ purified from yeast and is therefore useful for biochemical characterization of the interactions of pol ⑀ with other replication, recombination, and repair proteins. Identification and characterization of a proliferating cell nuclear antigen consensus interaction domain on Pol2p indicates that the motif is dispensable for DNA replication but is important for methyl methanesulfonate damage-induced DNA repair. Analysis of the putative zinc finger domain of Pol2p for zinc binding capacity demonstrates that it binds zinc. Mutations of the conserved cysteines in the putative zinc finger domain reduced zinc binding, indicating that cysteine ligands are directly involved in binding zinc. DNA polymerase epsilon (pol ⑀)1 purifies from yeast as a four-subunit holoenzyme. The subunits are Pol2p (256 kDa), Dpb2p (80 kDa), Dpb3p (34 kDa), and Dpb4p (29 kDa), and the genes encoding all four have been identified (1-5). Pol2p encodes the polymerase catalytic activity and is essential for the viability of yeast. Unlike the other essential polymerases, the polymerization function of pol ⑀ can actually be performed by another polymerase during DNA replication because deletion of the catalytic portion of the protein is not lethal (6). However, a point mutation in the polymerase active site is lethal, suggesting that normally the polymerase does function during DNA replication and that the presence of an inactive molecule is inhibitory. In Xenopus, pol ⑀ is also essential for DNA replication (7). Our previous work showed that Pol2p and Dpb2p expressed in insect cells form a heterodimer that itself forms a tetramer (Pol2p/Dpb2p) 2 mediated by self-interaction of the Dpb2p subunits. In addition, we found that Dpb3 and Dpb4 form a dimer that can interact with Pol2p/Dpb2p heterodimers. We identified a motif in the COOH-terminal, zinc finger domain of Pol2p (256 kDa) which was essential for interaction with Dpb2p and for dimerization, and we showed that this motif was essential for viability. The same region mediates interaction with Dpb3p/Dpb4p. Despite these advances in studying pairwise interactions and even interactions between the two heterodimers, Pol2p/Dpb2p and Dpb3p/Dpb4p, we were not able to reconstitute a holoenzyme containing all four subunits with the same stoichiometry as the holoenzyme isolated from y...