4DOC

Ternary complex of dna polymerase beta with a dideoxy terminated primer and 2'-deoxyguanosine 5'-beta, gamma-monochlororomethylene triphosphate:binding of S-isomer

4DOC の概要

• エントリーDOI: 10.2210/pdb4doc/pdb
• 関連するPDBエントリー: 4DO9 4DOA 4DOB
• 分子名称: DNA polymerase beta, C C G A C C G C G C A T C A G C, G C T G A T G C G (DOC), ... (9 entities in total)
• 機能のキーワード: stereoselectivity, polymerase, dna polymerase, transferase-dna complex, transferase/dna
• 由来する生物種: Homo sapiens (human); 詳細
• 細胞内の位置: Nucleus: P06746
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 48462.04

構造登録者

Batra, V.K. (登録日: 2012-02-09, 公開日: 2012-06-13, 最終更新日: 2023-09-13)

主引用文献

Wu, Y.,Zakharova, V.M.,Kashemirov, B.A.,Goodman, M.F.,Batra, V.K.,Wilson, S.H.,McKenna, C.E.
Beta,gamma-CHF- and beta,gamma-CHCl-dGTP Diastereomers: Synthesis, Discrete (31)P NMR Signatures, and Absolute Configurations of New Stereochemical Probes for DNA Polymerases
J.Am.Chem.Soc., 134:8734-8737, 2012

PubMed Abstract: Deoxynucleoside 5'-triphosphate analogues in which the β,γ-bridging oxygen has been replaced with a CXY group are useful chemical probes to investigate DNA polymerase catalytic and base-selection mechanisms. A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is nonequivalent (X ≠ Y). We report here a general solution to this long-standing problem with four examples of β,γ-CXY dNTP diastereomers: (S)- and (R)-β,γ-CHCl-dGTP (12a-1/12a-2) and (S)- and (R)-β,γ-CHF-dGTP (12b-1/12b-2). Central to their preparation was conversion of the prochiral parent bisphosphonic acids to the P,C-dimorpholinamide derivatives 7 of their (R)-mandelic acid monoesters, which provided access to the individual diastereomers 7a-1, 7a-2, 7b-1, and 7b-2 by preparative HPLC. Selective acidic hydrolysis of the P-N bond then afforded "portal" diastereomers, which were readily coupled to morpholine-activated dGMP. Removal of the chiral auxiliary by H(2) (Pd/C) gave the four individual diastereomeric nucleotides 12, which were characterized by (31)P, (1)H, and (19)F NMR spectroscopy and by mass spectrometry. After treatment with Chelex-100 to remove traces of paramagnetic ions, at pH ~10 the diastereomer pairs 12a,b exhibit discrete P(α) and P(β)(31)P resonances. The more upfield P(α) and more downfield P(β) resonances (and also the more upfield (19)F NMR resonance in 12b) are assigned to the R configuration at the P(β)-CHX-P(γ) carbons on the basis of the absolute configurations of the individual diastereomers as determined from the X-ray crystallographic structures of their ternary complexes with DNA and polymerase β.

PubMed: 22397499
DOI: 10.1021/ja300218x

実験手法

X-RAY DIFFRACTION (1.949 Å)