title: DNA base excision repair of uracil residues in reconstituted nucleosome core particles.
abstract: The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site-specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil-DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3- to 9-fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP-endonuclease 1, DNA polymerase beta and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil-DNA glycosylases and DNA polymerase beta on nucleosome cores.
authors.affiliation: Mutagenesis and Chromosome Dynamics Laboratories, Clare Hall Laboratories, Cancer Research UK. Hilde.Nilsen@cancer.org.uk
authors.forename: Hilde
authors.initials: H
authors.lastname: Nilsen
authors.forename: Tomas
authors.initials: T
authors.lastname: Lindahl
authors.forename: Alain
authors.initials: A
authors.lastname: Verreault
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004260:DNA Repair; D004275:DNA, Ribosomal / Q000235:genetics; D004447:Echinodermata / Q000235:genetics; D009707:Nucleosomes / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000235:genetics; D014498:Uracil / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D004275:DNA, Ribosomal; D009707:Nucleosomes; D012341:RNA, Ribosomal, 5S; D014498:Uracil