ジャーナル: Nat Commun / 年: 2017 タイトル: Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme. 著者: Jiangyu Yan / Thomas R Beattie / Adriana L Rojas / Kelly Schermerhorn / Tamzin Gristwood / Jonathan C Trinidad / Sonja V Albers / Pietro Roversi / Andrew F Gardner / Nicola G A Abrescia / Stephen D Bell / 要旨: Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family ...Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.
温度: 294.15 K / 手法: 蒸気拡散法, シッティングドロップ法 / pH: 7.5 詳細: OD280=17.7 mg/ml in 20 mM HEPES pH 7.5, 0.3 M NaCl, 1 mM MgCl2 and 1 mM b-ME mixed with 0.1 M Potassium thiocyanate and 30% w/v Polyethylene glycol monomethyl ether 2000 (Hampton Index)