The Second-Largest Subunit of the Mouse DNA Polymerase α-Primase Complex Facilitates Both Production and Nuclear Translocation of the Catalytic Subunit of DNA Polymerase α

Abstract

DNA polymerase ␣-primase is a replication enzyme necessary for DNA replication in all eukaryotes examined so far. Mouse DNA polymerase ␣ is made up of four subunits, the largest of which is the catalytic subunit with a molecular mass of 180 kDa (p180). This subunit exists as a tight complex with the second-largest subunit (p68), whose physiological role has remained unclear up until now. We set out to characterize these subunits individually or in combination by using a cDNA expression system in cultured mammalian cells. Coexpression of p68 markedly increased the protein level of p180, with the result that ectopically generated DNA polymerase activity was dramatically increased. Immunofluorescence analysis showed that while either singly expressed p180 or p68 was localized in the cytoplasm, cotransfection of both subunits resulted in colocalization in the nucleus. We identified a putative nuclear localization signal for p180 (residues 1419 to 1437) and found that interaction with p68 is essential for p180 to translocate into the nucleus. These results indicate that association of p180 with p68 is important for both protein synthesis of p180 and translocation into the nucleus, implying that p68 plays a pivotal role in the newly synthesized DNA polymerase ␣ complex.Chromosomal DNA replication in eukaryotes is a highly regulated process that requires a large number of replication factors, including distinct three types of DNA polymerases, ␣, ␦, and ε. Studies on the in vitro replication of simian virus 40 (SV40) DNA have made possible the functional identification of several DNA replication factors, including DNA polymerases (4, 17, 35). To date, DNA polymerase ␣-primase has been considered to provide RNA-DNA primers for the initiation of leading-strand synthesis and Okazaki fragment synthesis on the lagging strand during SV40 DNA replication (36,43,44).Mouse DNA polymerase ␣-primase complex was isolated from FM3A cells as a protein complex consisting of four subunits. The apparent molecular masses of these subunits based on their migration on sodium dodecyl sulfate (SDS) polyacrylamide gels are 180, 68, 54, and 46 kDa (39,40). The two smaller subunits, p46 and p54, can be dissociated from the other subunits by treatment with 50% ethylene glycol (38) and possess DNA primase activity, as demonstrated by the synthesis of unit-length oligoribonucleotides (7, 27). The largest subunit, p180, is the catalytic subunit of DNA polymerase and exhibits intrinsic DNA polymerase activity, as revealed in the baculovirus expression system (6). When the human p180 subunit is expressed alone in insect cells, it displays similarities to the holoenzyme such as an identical K m for the primer-template and deoxynucleoside triphosphate and similarities with respect to sensitivity to inhibitors, thermostability, DNA synthetic processivity, and fidelity (6). In contrast, the function of the second-largest subunit, p68, which is tightly bound to p180, remains unclear. To date, no enzymatic activity has been found for p68, and biochemical a...

Human Mcm10 Regulates the Catalytic Subunit of DNA Polymerase-α and Prevents DNA Damage during Replication

“…Although we do not yet understand the molecular mechanism underlying the association between Mcm10 and p180, our studies firmly establish that Mcm10 is required to stabilize p180 and that this occurs independently of p68 (Figure 1). This is further supported by the finding that p68 has no effect on the half-life of overexpressed p180 in mammalian cells although p68 facilitates the nuclear localization of p180 (Mizuno et al, 1998(Mizuno et al, , 1999, and the same is true for the homologues in budding yeast . Clearly, future studies are needed to explore how exactly Mcm10 contributes to replication fork progression and maintains genome integrity.…”

Species Specificity of Simian Virus 40 DNA Replication in Vitro Requires Multiple Functions of Human DNA Polymerase α

“…Alternatively, because p68 itself binds SV40 TAg (32,34,56), active complex formation may require a particular coordination between the interactions of p180 and p68 with TAg, which might be disturbed when the two subunits are derived from different species. Abundant evidence exists that p68 acts as a regulator of DNA polymerase ␣-primase (36,(55)(56)(57)(58), but the suppression of Mp180 C terminus-induced inhibition of SV40 DNA replication by Mp68 is only partial and is not evident in complexes such as (H671M)MH 2 and M 2 H 2 ( Fig. 6A and Ref.…”

“…This part of the polypeptide contains the DNAbinding domain necessary for interaction with the replication template but is also involved in interactions with the p68 subunit, which has no known catalytic activity but induces a conformational change into p180 (1,2,51,55). We investigated whether the inhibitory effect of the murine p180 C terminus on SV40 replication could be (in part) the consequence of a suboptimal interaction between murine p180 and human p68.…”

Quality Control of DNA Polymerase α

“…To examine interactions among the four subunits, we transfected four subunits in various combinations, and tested interactions of co-expressed subunits by coimmunoprecipitation analysis (summarized as a cartoon in Fig. 2; Mizuno et al 1998Mizuno et al , 1999. A crucial tool to express different combinations of subunits from a single plasmid was the introduction of IRES (Interal Ribosomal Entry Site) elements, which allowed analysis of subunit interdependency concerning protein expression and subcellular localisation (Fig.…”

“…The p46 protein, which is coupled to p180 by p54, synthesizes RNA primers and is involved in regulating their length; it also functions in cell cycle checkpoints (Arezi and Kuchta 2000). The 68 kDa subunit (p68) plays a crucial regulatory role in the early stage of chromosomal replication in yeast and has been shown to be essential for the nuclear import of p180 in mouse cells (Foiani et al 1994, Mizuno et al 1998. In this review, we discuss several layers of control that exist to ensure proper functioning of DNA polymerase α in the cell: regulation of transcription via cis-and trans-acting elements (part 2), ordered assembly of its four subunits into a functional complex (part 3.1), regulation of nuclear import (part 3.2), co-translational regulation (part 3.3), and quality control mechanisms to exclude aberrant proteins from acting in the nucleus (part 4).…”