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Deoxyribonucleic acid (DNA) repair and mutagenesis

Cells from patients with Cockayne syndrome (CS) are hypersensitive to DNA-damaging agents and are unable to restore damage-inhibited RNA synthesis. On the basis of repair kinetics of different types of lesions in transcriptionally active genes, we hypothesized previously that impaired transcription in CS cells is a consequence of defective transcription initiation after DNA damage induction. Here, we investigated the effect of UV irradiation on transcription by using an in vitro transcription system that allowed uncoupling of initiation from elongation events. Nuclear extracts prepared from UV-irradiated or mock-treated normal human and CS cells were assayed for transcription activity on an undamaged b-globin template. Transcription activity in nuclear extracts closely mimicked kinetics of transcription in intact cells: extracts from normal cells prepared 1 h after UV exposure showed a strongly reduced activity, whereas transcription activity was fully restored in ex-tracts prepared 6 h after treatment. Extracts from CS cells exhibited reduced transcription activity at any time after UV exposure. Reduced transcription activity in extracts coincided with a strong reduction of RNA polymerase II (RNAPII) containing hypophosphorylated C-terminal domain, the form of RNAPII known to be recruited to the initiation complex. These results suggest that inhibition of transcription after UV irradiation is at least partially caused by repression of transcription initiation and not solely by blocked elongation at sites of lesions. Generation of hypophosphorylated RNAPII after DNA damage appears to play a crucial role in restoration of transcription. CS proteins may be required for this process in a yet unknown way.

We also investigated the assembly of the nucleotide excision repair (NER) complex in normal and repair-deficient (xeroderma pigmentosum) human cells, employing a novel technique of local UV-irradiation combined with fluorescent antibody labeling. The damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors in the pre-incision complex including transcription-repair factor TFIIH. XPA associates relatively late, is required for anchoring of ERCC1-XPF and may be essential for activation of the endonuclease activity of XPG. These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight in the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of repair proteins at the site of the damage rather than a pre-assembled repair some.

References:
D.A.P. Rockx, R. Mason, A. van Hoffen, M. Craig Barton, E. Citterio, D.B. Bregman, A.A. van Zeeland, H. Vrieling, and L.H.F. Mullenders, UV-Induced inhibition of transcription involves repression of transcription initiation and phosphorylation of RNA polymerase II. Proc. Natl. Acad. Sci (USA) 97 (2000) 10503-10508.

M. Volker, M.J. Mon?, P. Karmakar, A. van Hoffen, W. Schul, W. Vermeulen, J.H.J. Hoeijmakers, R. van Driel, A.A. van Zeeland, and L.H.F. Mullenders, Sequential assembly of the nucleotide excision repair factors in vivo. Molecular Cell 8 (2001) 213-224.

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Record Number: 11210 / Last updated on: 2001-08-08
Collaboration sought: Information exchange/Training
Stage of development: Intermediate design, research phase
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