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Search results for: Mismatched Uracil Glycosylase Enzyme Buffer

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#17576692   2007/06/18 To Up

Different structural states in oligonucleosomes are required for early versus late steps of base excision repair.

Chromatin in eukaryotic cells is folded into higher order structures of folded nucleosome filaments, and DNA damage occurs at all levels of this structural hierarchy. However, little is known about the impact of higher order folding on DNA repair enzymes. We examined the catalytic activities of purified human base excision repair (BER) enzymes on uracil-containing oligonucleosome arrays, which are folded primarily into 30 nm structures when incubated in repair reaction buffers. The catalytic activities of uracil DNA glycosylase (UDG) and apyrimidinic/apurinic endonuclease (APE) digest G:U mismatches to completion in the folded oligonucleosomes without requiring significant disruption. In contrast, DNA polymerase beta (Pol beta) synthesis is inhibited in a major fraction ( approximately 80%) of the oligonucleosome array, suggesting that single strand nicks in linker DNA are far more accessible to Pol beta in highly folded oligonucleosomes. Importantly, this barrier in folded oligonucleosomes is removed by purified chromatin remodeling complexes. Both ISW1 and ISW2 from yeast significantly enhance Pol beta accessibility to the refractory nicked sites in oligonucleosomes. These results indicate that the initial steps of BER (UDG and APE) act efficiently on highly folded oligonucleosome arrays, and chromatin remodeling may be required for the latter steps of BER in intact chromatin.
Shima Nakanishi, Rajendra Prasad, Samuel H Wilson, Michael Smerdon

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