Journal article
Suppressed catalytic activity of base excision repair enzymes on rotationally positioned uracil in nucleosomes
Proceedings of the National Academy of Sciences - PNAS, Vol.100(13), pp.7465-7470
06/24/2003
Handle:
https://hdl.handle.net/2376/104468
PMCID: PMC164609
PMID: 12799467
Abstract
The majority of DNA in eukaryotic cells exists in the highly condensed structural hierarchy of chromatin, which presents a challenge to DNA repair enzymes in that recognition, incision, and restoration of the original sequence at most sites must take place within these structural constraints. To test base excision repair (BER) activities on chromatin substrates, an
in vitro
system was developed that uses human uracil DNA glycosylase (UDG), apyrimidinic/apurinic endonuclease (APE), and DNA polymerase β (pol β) on homogeneously damaged, rotationally positioned DNA in nucleosomes. We find that UDG and APE carry out their combined catalytic activities with reduced efficiency on nucleosome substrates (≈10% of that on naked DNA). Furthermore, these enzymes distinguish between two different rotational settings of the lesion on the histone surface, showing a 2- to 3-fold difference in activity between uracil facing ``toward'' and ``away from'' the histones. However, UDG and APE will digest such substrates to completion in a concentration-dependent manner. Conversely, the synthesis activity of pol β is inhibited completely by nucleosome substrates and is independent of enzyme concentration. These results suggest that the first two steps of BER, UDG and APE, may occur ``unassisted'' in chromatin, whereas downstream factors in this pathway (i.e., pol β) may require nucleosome remodeling for efficient DNA BER in at least some regions of chromatin in eukaryotic cells.
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Details
- Title
- Suppressed catalytic activity of base excision repair enzymes on rotationally positioned uracil in nucleosomes
- Creators
- Brian C Beard - Department of Biochemistry and Biophysics, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660; andSamuel H Wilson - Department of Biochemistry and Biophysics, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660; andMichael J Smerdon - Department of Biochemistry and Biophysics, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660; and
- Publication Details
- Proceedings of the National Academy of Sciences - PNAS, Vol.100(13), pp.7465-7470
- Academic Unit
- Molecular Biosciences, School of
- Publisher
- National Academy of Sciences
- Identifiers
- 99900546971101842
- Language
- English
- Resource Type
- Journal article