Synthesis and Nucleosome Structure of DNA Containing a UV Photoproduct at a Specific Site
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- 作者：Joseph V. Kosmoski and Michael J. Smerdon
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：July 20, 1999
- 年：1999
- 卷：38
- 期：29
- 页码：9485 - 9494
- 全文大小：148K
- 年卷期：v.38,no.29(July 20, 1999)
- ISSN：1520-4995
文摘
A strategy was developed to assemble nucleosomes specifically damaged at only one site andone structural orientation. The most prevalent UV photoproduct, a cis-syn cyclobutane thymine dimer (csCTD), was chemically synthesized and incorporated into a 30 base oligonucleotide harboring theglucocorticoid hormone response element. This oligonucleotide was assembled into a 165 base pair doublestranded DNA molecule with nucleosome positioning elements on each side of the cs CTD-containinginsert. Proton NMR verified that the synthetic photoproduct is the cis-syn stereoisomer of the CTD.Moreover, two different pyrimidine dimer-specific endonucleases cut ~90% of the dsDNA molecules.This cleavage is completely reversed by photoreactivation with E. coli UV photolyase, further demonstratingthe correct stereochemistry of the photoproduct. Nucleosomes were reconstituted by histone octamerexchange from chicken erythocyte core particles, and contained a unique translational and rotational settingof the insert on the histone surface. Hydroxyl radical footprinting demonstrates that the minor groove atthe cs CTD is positioned away from the histone surface about 5 bases from the nucleosome dyad.Competitive gel-shift analysis indicates there is a small increase in histone binding energy required forthe damaged fragment (
ages/gifchars/Delta.gif" BORDER=0 >ages/gifchars/Delta.gif" BORDER=0 >G ~ 0.15 kcal/mol), which does not prevent complete nucleosome loadingunder our conditions. Finally, folding of the synthetic DNA into nucleosomes dramatically inhibits cleavageat the cs CTD by T4 endonuclease V and photoreversal by UV photolyase. Thus, specifically damagednucleosomes can be experimentally designed for in vitro DNA repair studies.