ジャーナル: Proc Natl Acad Sci U S A / 年: 2011 タイトル: Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex. 著者: Kouta Mayanagi / Shinichi Kiyonari / Hirokazu Nishida / Mihoko Saito / Daisuke Kohda / Yoshizumi Ishino / Tsuyoshi Shirai / Kosuke Morikawa / 要旨: DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This ...DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This replication enzyme consists of the polymerase and exonuclease moieties responsible for DNA synthesis and editing (proofreading), respectively. Because of the editing activity, this enzyme ensures the high fidelity of DNA replication. However, it remains unclear how the PCNA-complexed enzyme temporally switches between the polymerizing and editing modes. Here, we present the three-dimensional structure of the Pyrococcus furiosus DNA polymerase B-PCNA-DNA ternary complex, which is the core component of the replisome, determined by single particle electron microscopy of negatively stained samples. This structural view, representing the complex in the editing mode, revealed the whole domain configuration of the trimeric PCNA ring and the DNA polymerase, including protein-protein and protein-DNA contacts. Notably, besides the authentic DNA polymerase-PCNA interaction through a PCNA-interacting protein (PIP) box, a novel contact was found between DNA polymerase and the PCNA subunit adjacent to that with the PIP contact. This contact appears to be responsible for the configuration of the complex specific for the editing mode. The DNA was located almost at the center of PCNA and exhibited a substantial and particular tilt angle against the PCNA ring plane. The obtained molecular architecture of the complex, including the new contact found in this work, provides clearer insights into the switching mechanism between the two distinct modes, thus highlighting the functional significance of PCNA in the replication process.
ダウンロード / ファイル: emd_5220.map.gz / 形式: CCP4 / 大きさ: 670.9 KB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
This is a map of PolB-PCNA-DNA complex
ボクセルのサイズ
X=Y=Z: 3.1 Å
密度
表面レベル
登録者による: 2.25 / ムービー #1: 2.25
最小 - 最大
-3.8578527 - 8.5686512
平均 (標準偏差)
-0.00000002 (±1.0)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
-9
-9
-9
サイズ
56
56
56
Spacing
56
56
56
セル
A=B=C: 173.59999 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
3.1
3.1
3.1
M x/y/z
56
56
56
origin x/y/z
0.000
0.000
0.000
length x/y/z
173.600
173.600
173.600
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-99
-99
-99
NX/NY/NZ
200
200
200
MAP C/R/S
1
2
3
start NC/NR/NS
-9
-9
-9
NC/NR/NS
56
56
56
D min/max/mean
-3.858
8.569
-0.000
-
添付データ
-
試料の構成要素
-
全体 : Pyrococcus furiosus PolB-PCNA-DNA
全体
名称: Pyrococcus furiosus PolB-PCNA-DNA
要素
試料: Pyrococcus furiosus PolB-PCNA-DNA
タンパク質・ペプチド: DNA polymerase
タンパク質・ペプチド: proliferating cell nuclear antigen
DNA: DNA
-
超分子 #1000: Pyrococcus furiosus PolB-PCNA-DNA
超分子
名称: Pyrococcus furiosus PolB-PCNA-DNA / タイプ: sample / ID: 1000 / 詳細: The sample was monodisperse 集合状態: One PolB binds to one trimer of PCNA and primed DNA Number unique components: 3