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

Christian S Eichinger,Takeshi Mizuno,Keiko Mizuno,Yasuyuki Miyake,Ken-Ichiro Yanagi,Naoko Imamoto,Fumio Hanaoka

doi:10.1074/jbc.m109.024760

Abstract

DNA polymerase alpha 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 alpha subunit, p180, the temperature-sensitive mouse cell line tsFT20 exhibits heat-labile DNA polymerase alpha 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 alpha, 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 alpha.

Highlights

The highly conserved DNA polymerase ␣-primase complex is the only eukaryotic polymerase that can initiate DNA synthesis de novo
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 [1,2,3,4]
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]

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction—Expression plasmids encoding p180 or p68 were previously described [14]. The tsFT20 cells were incubated at permissive (33 °C) or restrictive (39.5 °C) temperatures for the indicated times, harvested by centrifugation, and resuspended in SDS sample buffer. The remaining pellet was washed with CSK/Triton X-100 buffer, dissolved in 30 ␮l of SDS sample buffer, and subjected to SDS-PAGE and Western blot analysis using polyclonal anti-p180 antibody and monoclonal anti-polyubiquitin antibody. To define whether the disappearance of endogenous p180tsFT20 from the nucleus is dependent on nuclear export or the ubiquitin-mediated proteasome pathway, tsFT20 cells were incubated in the presence or absence of the nuclear export inhibitor LMB or the proteasome inhibitor MG132, and protein levels of p180tsFT20 were quantified by Western blot. ATT-3Ј; si68-3, 5Ј-GAUUCAGCCGAGUCCUUAATT-3Ј; uitin-mediated proteasome pathway and may be in si68-4, 5Ј-CAGAGACAUUGUUUCUAUATT-3Ј

RESULTS

Findings

DISCUSSION