ジャーナル: Elife / 年: 2021 タイトル: Structure and dynamics of the chromatin remodeler ALC1 bound to a PARylated nucleosome. 著者: Luka Bacic / Guillaume Gaullier / Anton Sabantsev / Laura C Lehmann / Klaus Brackmann / Despoina Dimakou / Mario Halic / Graeme Hewitt / Simon J Boulton / Sebastian Deindl / 要旨: The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a ...The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a candidate drug target for cancer therapy as its loss confers synthetic lethality in homologous recombination-deficient cells. However, structure-based drug design and molecular analysis of ALC1 have been hindered by the requirement for PARylation and the highly heterogeneous nature of this post-translational modification. Here, we reconstituted an ALC1 and PARylated nucleosome complex modified in vitro using PARP2 and HPF1. This complex was amenable to cryo-EM structure determination without cross-linking, which enabled visualization of several intermediate states of ALC1 from the recognition of the PARylated nucleosome to the tight binding and activation of the remodeler. Functional biochemical assays with PARylated nucleosomes highlight the importance of nucleosomal epitopes for productive remodeling and suggest that ALC1 preferentially slides nucleosomes away from DNA breaks.
タンパク質・ペプチド: Chromodomain-helicase-DNA-binding protein 1-like
タンパク質・ペプチド: Histone H3.2
タンパク質・ペプチド: Histone H4
タンパク質・ペプチド: Histone H2A type 1
タンパク質・ペプチド: Histone H2B 1.1
DNA: DNA (149-MER) Widom 601 sequence
DNA: DNA (149-MER) Widom 601 sequence
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超分子 #1: ALC1/CHD1L bound to a PARylated nucleosome
超分子
名称: ALC1/CHD1L bound to a PARylated nucleosome / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: all 詳細: The nucleosome was PARylated by PARP2 and HPF1 before addition of ALC1.
由来(天然)
生物種: Xenopus laevis (アフリカツメガエル)
分子量
理論値: 305 KDa
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分子 #1: Chromodomain-helicase-DNA-binding protein 1-like
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK IV 詳細: 3 uL were applied on grid and immediately blotted for 2.5 s at blot force 0..
フィルム・検出器のモデル: GATAN K3 BIOQUANTUM (6k x 4k) 撮影したグリッド数: 1 / 実像数: 26747 / 平均露光時間: 2.2 sec. / 平均電子線量: 45.0 e/Å2 / 詳細: Total dose was fractionated over 40 movie frames.
モデルのタイプ: INSILICO MODEL In silico モデル: Synthetic map at 30 A resolution generated from PDB entry 3LZ0 (free nucleosome) with the molmap command in ChimeraX.
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3.1.1)
最終 角度割当
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: cryoSPARC (ver. 3.2.0) / 詳細: Non-uniform refinement in cryoSPARC.
最終 3次元分類
クラス数: 5 / ソフトウェア - 名称: cryoSPARC (ver. 3.2.0) 詳細: The final set of 5487 particles used for non-uniform refinement in cryoSPARC were identified by GMM clustering of results from 3D variability analysis in cryoSPARC.