Nucleic Acids Research, 2001, Vol. 29, No. 7 1574-1581
© 2001 Oxford University Press
Different dynamics in nuclear entry of subunits of the repair/transcription factor TFIIH
Istituto di Genetica Biochimica ed Evoluzionistica del CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
We report here the different ways in which four subunits of the basal transcription/repair factor TFIIH (XPB, XPD, p62 and p44) and the damage recognition XPC repair protein can enter the nucleus. We examined their nuclear localization by transiently expressing the gene products tagged with the enhanced green fluorescent protein (EGFP) in transfected 3T3 cells. In agreement with the identification of more than one putative nuclear localization signal (NLS) in their protein sequences, XPB, XPC, p62 and p44 chimeras were rapidly sorted to the nucleus. In contrast, the XPD–EGFP chimeras appeared mainly localized in the cytoplasm, with a minor fraction of transfectants showing the EGFP-based fluorescence also in the nucleus. The ability of the XPD chimeras to enter the nucleus was confirmed by western blotting on fractionated cell extracts and by functional complementation of the repair defect in the UV5 rodent cells, mutated in the XPD homologous gene. By deletion mutagenesis, we were unable to identify any sequence specific for nuclear localization. In particular, deletion of the putative NLS failed to affect subcellular localization and, conversely, the C-terminal part of XPD containing the putative NLS showed no specific nuclear accumulation. These findings suggest that the nuclear entry of XPD depends on its complexation with other proteins in the cytoplasm, possibly other components of the TFIIH complex.
* To whom correspondence should be addressed. Tel: +39 0382 546325; Fax: +39 0382 422286; Email: pedrini{at}igbe.pv.cnr.it Present address: Fabio Santagati, GSF-Forschungszentrum, Institute of Mammalian Genetics, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany
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