Aberrant DNA Polymerase α Is Excluded from the Nucleus by Defective Import and Degradation in the Nucleus

Abstract

DNA polymerase ␣ is essential for the onset of eukaryotic DNA replication. Its correct folding and assembly within the nuclear replication pre-initiation complex is crucial for normal cell cycle progression and genome maintenance. Due to a single point mutation in the largest DNA polymerase ␣ subunit, p180, the temperature-sensitive mouse cell line tsFT20 exhibits heatlabile DNA polymerase ␣ activity and S phase arrest at restrictive temperature. In this study, we show that an aberrant form of endogenous p180 in tsFT20 cells (p180 tsFT20 ) is strictly localized in the cytoplasm while its wild-type counterpart enters the nucleus. Time-lapse fluorescence microscopy with enhanced green fluorescent protein-tagged or photoactivatable green fluorescent protein-tagged p180 tsFT20 variants and inhibitor analysis revealed that the exclusion of aberrant p180 tsFT20 from the nucleus is due to two distinct mechanisms: first, the inability of newly synthesized (cytoplasmic) p180 tsFT20 to enter the nucleus and second, proteasome-dependent degradation of nuclear-localized protein. The nuclear import defect seems to result from an impaired association of aberrant de novo synthesized p180 tsFT20 with the second subunit of DNA polymerase ␣, p68. In accordance, we show that RNA interference of p68 results in a decrease of the overall p180 protein level and in a specific increase of cytoplasmic localized p180 in NIH3T3 cells. Taken together, our data suggest two mechanisms that prevent the nuclear expression of aberrant DNA polymerase ␣.The highly conserved DNA polymerase ␣-primase complex is the only eukaryotic polymerase that can initiate DNA synthesis de novo. Thus, its recruitment is a crucial step in the tightly regulated stepwise assembly of the replication machinery in eukaryotic cells. This complex is required for the synthesis of RNA primers, an essential prerequisite for the initiation of replication, and for the discontinuous synthesis of Okazaki fragments on the lagging strand (1-4). Moreover, DNA polymerase ␣ plays a fundamental role in coordinating DNA replication, DNA repair, and cell cycle progression (1), in telomere capping and length regulation (5-9), and in the epigenetic control of transcriptional silencing and nucleosome reorganization (10, 11).The DNA polymerase ␣-primase complex consists of four subunits, each of which is conserved in eukaryotes; in yeast, all four subunits are essential for viability (2). The largest subunit, 180 kDa (p180), harbors the catalytic polymerase ␣ activity. The two smallest subunits, 54 kDa (p54) and 46 kDa (p46), provide primase activity. 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 (12). 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 (13,14).DNA replication takes place during a restricted period in the cell cycle, in ...

Structural Basis for the Interaction of a Hexameric Replicative Helicase with the Regulatory Subunit of Human DNA Polymerase α-Primase

“…The p180 DNA polymerase subunit then elongates the RNA primers into RNA/ DNA primers of about 30 -35 nucleotides. The p68 or B-subunit is not essential for the enzymatic activities of Pol-prim, but it is essential in vivo and may regulate Pol-prim function in response to cell cycle signaling or at telomeres (13)(14)(15)(16)(17). Structure-function studies indicate that the C-terminal zinc domain of p180 anchors both the p68 subunit and the p58 subunit of the primase dimer in the complex (9,18).…”

Structure of a DNA Polymerase α-Primase Domain That Docks on the SV40 Helicase and Activates the Viral Primosome

“…Its primase subunits (p48 and p58) initially synthesize an 8 -12-nucleotide RNA primer, which is then shifted internally to the active site of the associated p180 DNA polymerase subunit for extension into a 30 -35-nucleotide RNA-DNA primer (4,5). The fourth subunit of the pol-prim complex, known as p68 or B-subunit, lacks enzyme activity but is essential for S phase entry in yeast (6) and for p180 accumulation and nuclear import (7)(8)(9)(10). The sequence and structure of the C-terminal domain (CTD) of p68 are conserved in the B-subunits of DNA polymerases ␦ and ⑀; the p68CTD is tightly associated with the CTD of the p180 subunit (11-13) (see Fig.…”

Quality Control of DNA Polymerase α

“…Upon temperature shift to restrictive temperature, nuclear localized p180 tsFT20 GFP is rapidly removed from the nucleus, a process that can be blocked by addition of MG132, a specific proteasome inhibitor ( Fig. 7C; Eichinger, et al 2009). Thus, we concluded that aberrant p180 is degraded in the nucleus in a proteasome-dependent manner.…”

“…Second, nuclearlocalized protein is degraded in a proteasome-dependent manner in the nucleus and to a minor extent in the cytoplasm after export from the nucleus ( Fig. 8; Eichinger, et al 2009). Very recently it has been reported that misfolded nuclear proteins are degraded by a unique E3 ubiquitin ligase in S. cerevisiae, San1 (Gardner et al 2005;Rosenbaum JC, et al 2011).…”

“…Unexpectedly, our investigation using an aberrant mutant version of p180 (p180 tsFT20 ) and live cell imaging techniques allowed us to recognize novel protein quality control mechanisms acting on DNA polymerase α which ensure exclusion of aberrant forms of p180 from the nucleus (Eichinger et al 2009). In general, misfolded and non-functional proteins must be degraded to assure the correct functioning of cellular processes.…”