Identification of the NLS sequence in the ERCC5/XP-G protein

Journal Article · Sat Dec 31 04:00:00 EST 1994 · Environmental and Molecular Mutagenesis

OSTI ID:88886

Knauf, J A; Park, M S; MacInnes, M A ^[1]

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1. Los Alamos National Laboratory, NM (United States)

The Excision Repair Cross Complementing gene 5 (ERCC5) protein is required for nucleotide excision repair and is involved in the human disease xeroderma pigmentosum complementation group G. Here we show, by immunofluorescence with a rabbit antibody to the C-terminus of the ERCC5 protein, that the ERCC5 protein is located in the nucleus. The ERCC5 protein has a calculated molecular weight of 136.2 kDa, thus requiring a NLS signal to enter the nucleus. Sequence analysis of the ERCC5 protein indicated 4 putative NLS sites. To determine which of the 4 putative NLS sites is involved in nuclear localization of the ERCC5 protein, fragments of the ERCC5 gene were cloned into the vector pCH110 (Pharmacia). The ERCC5 fragments were cloned in frame with and upstream of the beta-galactosidase gene which is under the control of a mammalian promoter. The plasmid was then transfected into HeLa cells and the cellular location of the fusion protein was determined by histochemical staining with X-gal or by immunofluorescence using an antibody to the beta-galactosidase protein. The beta-galactosidase protein alone is predominately located in the cytosol. However, when the last 159 amino acids of the C-terminus are fused to the beta-galactosidase protein the fusion protein is found exclusively in the nucleus.

• DOE Contract Number: W-7405-ENG-36
• OSTI ID: 88886
• Report Number(s): CONF-9405324--
• Journal Information: Environmental and Molecular Mutagenesis, Journal Name: Environmental and Molecular Mutagenesis Journal Issue: Suppl.23 Vol. 23; ISSN 0893-6692; ISSN EMMUEG
• Country of Publication: United States
• Language: English

Related Subjects

55 BIOLOGY AND MEDICINE
BASIC STUDIES
AMINO ACID SEQUENCE
ANIMAL CELLS
DNA REPAIR
GENETIC CONTROL
PROTEINS
SKIN DISEASES