Analysis of the Essential Functions of the C-terminal Protein/Protein Interaction Domain of Saccharomyces cerevisiae pol ε and Its Unexpected Ability to Support Growth in the Absence of the DNA Polymerase Domain

Abstract

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...

p53 deficiency rescues apoptosis and differentiation of multiple cell types in zebrafish flathead mutants deficient for zygotic DNA polymerase δ1

“…The zinc-finger domains have been suggested to be important for internal stability of the protein. 1 Furthermore, in the case of mouse Pola1 44 and yeast Pold 45 and Pole, 46 these domains have been shown to be required for binding of the catalytic subunits to the B subunits (Cdc1/Pold2) of the holoenzymes. [44][45][46] Accordingly, mutational analyses in budding yeast revealed that mutations within the zinc-finger domains of Pold and Pole abolish polymerase function in vivo.…”

“…1 Furthermore, in the case of mouse Pola1 44 and yeast Pold 45 and Pole, 46 these domains have been shown to be required for binding of the catalytic subunits to the B subunits (Cdc1/Pold2) of the holoenzymes. [44][45][46] Accordingly, mutational analyses in budding yeast revealed that mutations within the zinc-finger domains of Pold and Pole abolish polymerase function in vivo. 46,47 In sum, given the importance of the domains lost in the different mutant versions of zebrafish Pold1, together with the indistinguishable phenotypic strengths of the three alleles, we suspect that they all represent pold1 null mutations.…”

“…[44][45][46] Accordingly, mutational analyses in budding yeast revealed that mutations within the zinc-finger domains of Pold and Pole abolish polymerase function in vivo. 46,47 In sum, given the importance of the domains lost in the different mutant versions of zebrafish Pold1, together with the indistinguishable phenotypic strengths of the three alleles, we suspect that they all represent pold1 null mutations.…”

The C-terminal zinc finger of the catalytic subunit of DNA polymerase is responsible for direct interaction with the B-subunit

“…In the case of mouse Pol a, an important role for the ZnF modules in binding to the B-subunit p68 been demonstrated (21) and the structure of one of the two ZnF modules recently solved (22). In Pol e, the extended C-terminal domain of the protein, which includes the ZnF modules, has also been implicated in B-subunit binding, as well as in Pol e dimerization and in checkpoint signalling (4).…”

The Quaternary Structure of DNA Polymerase ε from Saccharomyces cerevisiae

“…The largest subunit, Pol2p, contains the catalytic site and an essential putative zinc-finger domain at the C terminus (9). The other three subunits have an unknown function.…”