Aberrant DNA polymerase alpha is excluded from the nucleus by defective import and degradation in the nucleus.
J Biol Chem
; 284(44): 30604-14, 2009 Oct 30.
Article
em En
| MEDLINE
| ID: mdl-19726690
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Núcleo Celular
/
Transporte Ativo do Núcleo Celular
/
DNA Polimerase I
Limite:
Animals
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
Japão