DNA repair of a single UV photoproduct in a designed nucleosome.
Eukaryotic DNA repair enzymes must interact with the architectural hierarchy of chromatin. The challenge of finding damaged DNA complexed with histone proteins in nucleosomes is complicated by the need to maintain local chromatin structures involved in regulating other DNA processing events. The heterogeneity of lesions induced by DNA-damaging agents has led us to design homogeneously damaged substrates to directly compare repair of naked DNA with that of nucleosomes. Here we report that nucleotide excision repair in Xenopus nuclear extracts can effectively repair a single UV radiation photoproduct located 5 bases from the dyad center of a positioned nucleosome, although the nucleosome is repaired at about half the rate at which the naked DNA fragment is. Extract repair within the nucleosome is >50-fold more rapid than either enzymatic photoreversal or endonuclease cleavage of the lesion in vitro. Furthermore, nucleosome formation occurs (after repair) only on damaged naked DNA (165-bp fragments) during a 1-h incubation in these extracts, even in the presence of a large excess of undamaged DNA. This is an example of selective nucleosome assembly by Xenopus nuclear extracts on a short linear DNA fragment containing a DNA lesion.
Kosmoski
J V
JV
Biochemistry and Biophysics, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4660, USA.
Ackerman
E J
EJ
Smerdon
M J
MJ
D000818:Animals; D001483:Base Sequence; D004249:DNA Damage; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D005260:Female; D066298:In Vitro Techniques; D009707:Nucleosomes / Q000378:metabolism* / Q000528:radiation effects; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D010777:Photochemistry; D014466:Ultraviolet Rays; D014981:Xenopus
D009707:Nucleosomes; D009838:Oligodeoxyribonucleotides; D011088:DNA Ligases