Abstract
The influence of chromatin structure on DNA metabolic processes, including DNA replication and repair, has been a matter of intensive studies in recent years. Although the human mismatch repair (MMR) reaction has been reconstituted using purified proteins, the influence of chromatin structure on human MMR is unknown. This study examines the interaction between human MutSalpha and a mismatch located within a nucleosome or between two nucleosomes. The results show that, whereas MutSalpha specifically recognizes both types of nucleosomal heteroduplexes, the protein bound the mismatch within a nucleosome with much lower efficiency than a naked heteroduplex or a heterology free of histone proteins but between two nucleosomes. Additionally, MutSalpha displays reduced ATPase- and ADP-binding activity when interacting with nucleosomal heteroduplexes. Interestingly, nucleosomes block ATP-induced MutSalpha sliding along the DNA helix when the mismatch is in between two nucleosomes. These findings suggest that nucleosomes may inhibit MMR in eukaryotic cells. The implications of these findings for our understanding of eukaryotic MMR are discussed.
Highlights
The nucleosome is the basic structural unit of eukaryotic chromatin, and is comprised of an octamer of histone proteins wrapped with a DNA duplex of ϳ147 bp
Reconstitution and Characterization of Mismatch-containing Nucleosomes—Histone octamers were assembled from purified recombinant human histone proteins and purified by a fast-protein liquid chromatography Superdex 200 column and verified by SDS-gel electrophoresis (Fig. 1B)
To determine if the differences in ADP binding among reactions reflect changes in the ADP-binding ability of MSH2 or the binding of available ADP to MSH2 in Growing evidence supports a role for chromatin structure in mismatch repair (MMR)
Summary
The nucleosome is the basic structural unit of eukaryotic chromatin, and is comprised of an octamer of histone proteins wrapped with a DNA duplex of ϳ147 bp. Human MMR has been reconstituted with purified proteins in vitro using naked DNA heteroduplexes [13, 14], the influence of chromatin structure on human MMR has not been analyzed. It is not known how human MMR proteins recognize and interact with mismatches in nascent or damaged DNA. MutS␣ bound the mismatch with lower efficiency in histone octamer-bound DNA than in histone octamer-free DNA, and ATP-provoked sliding of MutS␣ along DNA helices is blocked when the mismatch is flanked on each side by two nucleosomes These observations strongly support a notion that chromatin remodeling and/or modification factors are required for MMR in eukaryotic cells
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