Endonuclease III interactions with DNA substrates. 2. The DNA repair enzyme endonuclease III binds differently to intact DNA and to apyrimidinic/apurinic DNA substrates as shown by tryptophan fluorescence quenching.
Endonuclease III interactions with DNA substrates. 2. The DNA repair enzyme endonuclease III binds differently to intact DNA and to apyrimidinic/apurinic DNA substrates as shown by tryptophan fluorescence quenching.
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核酸内切酶 III 与 DNA 底物相互作用。
DOI：
10.1021/bi00008a018
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Scholes,CP
中科院分区：
文献类型：
--
作者：
Xing,D;Dorr,R;Cunningham,RP;Scholes,CP
摘要：我们测量了DNA修复酶内切酶III的荧光，以发现未损伤DNA和AP（无嘧啶或无嘌呤）DNA对其色氨酸的扰动，并估计其与完整DNA和AP DNA的结合亲和力。内切酶III具有两个色氨酸，Trp132位于AP裂解酶活性位点附近可能具有柔韧性的螺旋-发夹-螺旋区域，Trp178位于内切酶III的含铁硫中心的区域；Trp132是一种更易溶于溶剂的色氨酸[j]。李建平，李建平，李建平，等（2002）中国科学：科学与发展(5),344 - 344。荧光发射峰值波长接近350 nm（激发波长为290 nm），表明荧光色氨酸暴露于极性环境中。碘化物猝灭色氨酸荧光表明确实存在两种色氨酸，它们对阴离子猝灭剂的可及性不同。用高分子量双相无损聚（dAdT）滴定内切酶III时发生显著（~ 60%）荧光猝灭。与poly （dAdT）的表观二级非特异性结合常数为4 × 107 M-1，每个内切酶III与poly （dAdT）的结合位点约有12个碱基对。这种与双链DNA的非特异性结合具有离子特性，并且没有单链DNA带来的荧光猝灭。高分子量双链DNA与未损伤双链19-mer寡核苷酸的荧光猝灭滴定结果表明，高分子量DNA与未损伤双链19-mer的结合常数约为未损伤双链19-mer的400倍。凝胶位移和足迹实验清楚地表明，含有中心AP或非催化还原AP位点的双寡核苷酸底物与内切酶HI强烈结合[O 'Handley， S., Scholes， CP, & Cunningham， R. P.(1995) Biochemistry 34, 2528-2536]，但在AP或还原AP寡核苷酸底物存在时，荧光猝灭减少。这种AP底物的特异性结合必须涉及荧光色氨酸-不同于非特异性结合到未损伤的双链DNA。这种差异可能与通过足迹测定的特异性ap中心结合位点的5到7个碱基对大小与通过这些荧光测量确定的非特异性结合位点的12个碱基对大小之间的差异有关。在与含有中心AP位点或还原AP位点的寡核苷酸19-mer底物的竞争中，高分子量poly (dAdT) DNA带来的色氨酸荧光猝灭被消除并减少到接近其未猝灭值。这种竞争性消除的定量表明，还原的AP 19-mer底物的特定结合常数为4 × 107 M-1，而AP 19-mer底物的结合常数远大于108 M-1。大肠杆菌核酸内切酶III (endo III) 1是一种DNA修复酶，具有DNA/ v -糖基化酶和无嘌呤/无嘧啶（AP）裂解酶活性（Katcher & Wal-lace, 1983; Doetsch & Cunningham, 1990）。作为一种AP裂解酶，该酶在发生碱基损失的AP位点引入单链断裂。细菌、酵母、牛和人类细胞共享一类保守的酶，它们与大肠杆菌的内切酶具有相似的分子量和相同的底物特异性，用于破坏DNA的切割（Doetsch et al., 1987）。这和同伴的总体目的有关
Revised Manuscript Received November 1, 1994® abstract: We have measured the fluorescence of the DNA repair enzyme endonuclease III to discover perturbation to its tryptophans by undamaged DNA and AP (apyrimidinic or apurinic) DNA and to estimate binding affinity for intact and AP DNAs. Endonuclease III has twotryptophans, Trp132 in a helix-hairpin—helix region of possible flexibility near the active site for AP lyase activity and Trp178 in the domain containing the iron—sulfur center of endonuclease III; Trp132 is the more solvent-accessible tryptophan [Kuo, C.-F., McRee, D. E., Fisher, C. L., O’Handley, S. F., & Cunningham, RP (1992) Science 258, 434—440]. The fluorescence emission peak wavelength near 350 nm (excitation at 290 nm) indicated an exposure of the fluorescing tryptophans to a polar environment. Quenching of tryptophan fluorescence by iodide demonstrated that there are indeed two tryptophans which are differently accessible to anionic quencher. Significant (~ 60%) fluorescence quenching occurred when endonuclease III was titrated with high molecular weightduplex undamaged poly (dAdT). The apparent second-order nonspecific binding constant to poly (dAdT) was 4 x 107 M-1, and there were approximately 12 base pairs per endonuclease III binding site for binding to poly (dAdT). This nonspecific binding to duplex DNA had ionic character, and there was no fluorescence quenching brought on by single-stranded DNA. A comparison between fluorescence quenching titrations of high molecular weight duplex DNA and undamaged duplex 19-mer oligonucleotide showed that the binding constant to the high molecular weight DNA was~ 400-fold larger thanto the undamaged 19-mer. Gel shift and footprinting experiments clearly show that duplex oligonucleotide substrates containing a central AP or noncatalyzable reduced AP site strongly bind to endonuclease HI [O’Handley, S., Scholes, CP, & Cunningham, R. P.(1995) Biochemistry 34, 2528—2536], but there was reduced fluorescence quenching in the presence of AP or reduced AP oligonucleotide substrates. Thespecific binding of such AP substrates must involve fluorescing tryptophan-(s) differently from nonspecific binding to undamaged duplex DNA. This difference may correlate with the difference between the five-to seven-base pair size of the specific, AP-centered binding site determined by footprinting (companion paper) and the 12-base pair size of the nonspecificbinding site determined from these fluorescence measurements. In competition with oligonucleotide 19-mer substrates containing either a central AP site or a reduced AP site, tryptophan fluorescence quenching brought on by high molecular weight poly (dAdT) DNA was eliminated and reduced to nearly its unquenched value. Quantitation of this competitive elimination indicated a specific binding constant of order 4 x 107 M-1 for the reduced AP 19-mersubstrate and a binding constant much greater than 108 M-1 for the AP 19-mer substrate.Endonuclease III (endo III) 1 from Escherichia coli is a DNA repairenzyme that has both DNA/V-glycosylase and apurinic/apyrimidinic (AP) lyase activities (Katcher & Wal-lace, 1983; Doetsch & Cunningham, 1990). As an AP lyase, the enzyme introduces a single-strand break at the AP site where loss of a base has occurred. Bacteria, yeast, bovine, and human cells share a conserved class of enzymes with similar molecular weights and the same substrate specificity as endo IH from E. coli for damaged DNA cleavage (Doetsch et al., 1987). The overall purpose of this and the companion