ジャーナル: PLoS Biol / 年: 2007 タイトル: Different quaternary structures of human RECQ1 are associated with its dual enzymatic activity. 著者: Laura Muzzolini / Fabienne Beuron / Ardan Patwardhan / Venkateswarlu Popuri / Sheng Cui / Benedetta Niccolini / Mathieu Rappas / Paul S Freemont / Alessandro Vindigni / 要旨: RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual ...RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and transmission electron microscopy show that the equilibrium between the two assembly states is affected by single-stranded DNA (ssDNA) and ATP binding, where ATP or ATPgammaS favors the smaller oligomeric form. Our three-dimensional electron microscopy reconstructions of human RECQ1 reveal a complex cage-like structure of approximately 120 A x 130 A with a central pore. This oligomeric structure is stabilized under conditions in which RECQ1 is proficient in strand annealing. In contrast, competition experiments with the ATPase-deficient K119R and E220Q mutants indicate that RECQ1 monomers, or tight binding dimers, are required for DNA unwinding. Collectively, our findings suggest that higher-order oligomers are associated with DNA strand annealing, and lower-order oligomers with DNA unwinding.
タイプ: NEGATIVE 詳細: RECQ1 incubated with 1.5 molar excess of ssDNA for 15 min, then adsorbed onto glow-discharged carbon-coated grids and negatively-stained with 1% uranyl acetate.
凍結
凍結剤: ETHANE
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電子顕微鏡法
顕微鏡
FEI/PHILIPS CM200FEG
アライメント法
Legacy - Electron beam tilt params: 0
日付
2005年6月6日
撮影
カテゴリ: FILM / フィルム・検出器のモデル: KODAK SO-163 FILM / デジタル化 - スキャナー: OTHER / デジタル化 - サンプリング間隔: 6.35 µm / ビット/ピクセル: 8