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

DNA polymerase α 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 α subu...

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Bibliographic Details
Published in:The Journal of biological chemistry 2009-10, Vol.284 (44), p.30604
Main Authors: Christian S. Eichinger, Takeshi Mizuno, Keiko Mizuno, Yasuyuki Miyake, Ken-ichiro Yanagi, Naoko Imamoto, Fumio Hanaoka
Format: Article
Language:English
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Summary:DNA polymerase α 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 α subunit, p180, the temperature-sensitive mouse cell line tsFT20 exhibits heat-labile DNA polymerase α 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 α, 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 α.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M109.024760