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首页> 美国卫生研究院文献>other >EXPRESSION AND BIOCHEMICAL CHARACTERIZATION OF THE PLASMODIUM FALCIPARUM DNA REPAIR ENZYME FLAP ENDONUCLEASE-1 (PfFEN-1)
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EXPRESSION AND BIOCHEMICAL CHARACTERIZATION OF THE PLASMODIUM FALCIPARUM DNA REPAIR ENZYME FLAP ENDONUCLEASE-1 (PfFEN-1)

机译：恶性疟原虫DNA修复酶Flap Endonuclease-1（PfFEN-1）的表达及生物化学特性

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Flap Endonuclease-1 (FEN-1) is a structure-specific endonuclease that is critical for the resolution of single-stranded DNA flap intermediates that form during long patch DNA Base Excision Repair (BER). This investigation reports that Plasmodium species encode FEN-1 homologs. Protein sequence analysis revealed the N and I domains of Plasmodium falciparum (PfFEN-1) and Plasmodium yoelii (PyFEN-1) to be homologous to FEN-1 from other species. However, each possessed an extended C domain which had limited homology to apicomplexan FEN-1s and no homology to eukaryotic FEN-1s. A conserved Proliferating Cell Nuclear Antigen (PCNA) binding site was identified at an internal location rather than the extreme C-terminal location typically seen in FEN-1 from other organisms. The endonuclease and exonuclease activities of PfFEN-1 and PyFEN-1 were investigated using recombinant protein produced in Escherichia coli. Pf and PyFEN-1 possessed DNA structure-specific flap endonuclease and 5′→3′ exonuclease activities, similar to FEN-1’s from other species. Endonuclease activity was stimulated by Mg+2 or Mn+2 and inhibited by monovalent ions (>20.0 mM). A PfFEN-1 C-terminal truncation mutant lacking the terminal 250 amino acids (PfFEN-1ΔC) had endonuclease activity that was ~130-fold greater (kcat= 1.2x10−1) than full-length PfFEN-1 (kcat= 9.1x10−4) or ~240-fold greater than PyFEN-1 (kcat= 4.9x10−4) in vitro. PfFEN-1 generated a nicked DNA substrate that was ligated by recombinant Pf DNA Ligase I (PfLigI) using an in vitro DNA repair assay. Plasmodium FEN-1s have enzymatic activities similar to other species but contain extended C-termini and a more internally located PCNA binding site.
机译：Flap Endonuclease-1（FEN-1）是一种结构特异性核酸内切酶，对于解决长补丁DNA碱基切除修复（BER）期间形成的单链DNA襟翼中间体至关重要。这项研究报告疟原虫物种编码FEN-1同源物。蛋白质序列分析显示，恶性疟原虫（PfFEN-1）和约氏疟原虫（PyFEN-1）的N和I结构域与其他物种的FEN-1同源。然而，每个人都具有扩展的C结构域，其与apicomplexan FEN-1s具有有限的同源性，而与真核生物的FEN-1没有同源性。在内部位置而不是在其他生物的FEN-1中通常看到的极端C端位置，发现了一个保守的增殖细胞核抗原（PCNA）结合位点。使用在大肠杆菌中产生的重组蛋白研究了PfFEN-1和PyFEN-1的核酸内切酶和核酸外切酶活性。 Pf和PyFEN-1具有特定于DNA结构的襟翼内切核酸酶和5'→3'核酸外切酶活性，与其他物种的FEN-1相似。核酸内切酶活性受Mg +2 或Mn +2 刺激，并被单价离子（> 20.0 mM）抑制。缺少末端250个氨基酸（PfFEN-1ΔC）的PfFEN-1 C端截短突变体的核酸内切酶活性比全长PfFEN高约130倍（kcat = 1.2x10 -1 ） -1（kcat = 9.1x10 −4 ）或比PyFEN-1（kcat = 4.9x10 −4 ）大240倍。 PfFEN-1产生了带切口的DNA底物，使用体外DNA修复测定法将其与重组Pf DNA连接酶I（PfLigI）连接。疟原虫FEN-1具有与其他物种相似的酶促活性，但含有扩展的C末端和位于内部的PCNA结合位点。

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