ジャーナル: Nature / 年: 2021 タイトル: Mechanism for Cas4-assisted directional spacer acquisition in CRISPR-Cas. 著者: Chunyi Hu / Cristóbal Almendros / Ki Hyun Nam / Ana Rita Costa / Jochem N A Vink / Anna C Haagsma / Saket R Bagde / Stan J J Brouns / Ailong Ke / 要旨: Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array. Spacer insertion is carried out by the Cas1-Cas2 integrase complex. A ...Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array. Spacer insertion is carried out by the Cas1-Cas2 integrase complex. A substantial fraction of CRISPR-Cas systems use a Fe-S cluster containing Cas4 nuclease to ensure that spacers are acquired from DNA flanked by a protospacer adjacent motif (PAM) and inserted into the CRISPR array unidirectionally, so that the transcribed CRISPR RNA can guide target searching in a PAM-dependent manner. Here we provide a high-resolution mechanistic explanation for the Cas4-assisted PAM selection, spacer biogenesis and directional integration by type I-G CRISPR in Geobacter sulfurreducens, in which Cas4 is naturally fused with Cas1, forming Cas4/Cas1. During biogenesis, only DNA duplexes possessing a PAM-embedded 3'-overhang trigger Cas4/Cas1-Cas2 assembly. During this process, the PAM overhang is specifically recognized and sequestered, but is not cleaved by Cas4. This 'molecular constipation' prevents the PAM-side prespacer from participating in integration. Lacking such sequestration, the non-PAM overhang is trimmed by host nucleases and integrated to the leader-side CRISPR repeat. Half-integration subsequently triggers PAM cleavage and Cas4 dissociation, allowing spacer-side integration. Overall, the intricate molecular interaction between Cas4 and Cas1-Cas2 selects PAM-containing prespacers for integration and couples the timing of PAM processing with the stepwise integration to establish directionality.
ダウンロード / ファイル: emd_23849.map.gz / 形式: CCP4 / 大きさ: 36.3 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
ボクセルのサイズ
X=Y=Z: 1.32 Å
密度
表面レベル
登録者による: 0.145 / ムービー #1: 0.145
最小 - 最大
-0.17644785 - 0.9397226
平均 (標準偏差)
-0.00034170382 (±0.029530449)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
0
0
0
サイズ
212
212
212
Spacing
212
212
212
セル
A=B=C: 279.84 Å α=β=γ: 90.0 °
CCP4マップ ヘッダ情報:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.32
1.32
1.32
M x/y/z
212
212
212
origin x/y/z
0.000
0.000
0.000
length x/y/z
279.840
279.840
279.840
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
0
0
0
NX/NY/NZ
376
376
376
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
212
212
212
D min/max/mean
-0.176
0.940
-0.000
-
添付データ
-
試料の構成要素
-
全体 : Half integration complex and Cas4 dissociated from PAM side
全体
名称: Half integration complex and Cas4 dissociated from PAM side
要素
複合体: Half integration complex and Cas4 dissociated from PAM side
タンパク質・ペプチド: half integration complex of Cas1-Cas2
-
超分子 #1: Half integration complex and Cas4 dissociated from PAM side
超分子
名称: Half integration complex and Cas4 dissociated from PAM side タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: all / 詳細: half integration in cas4 containing system