Hyperthermophilic enzyme APE1547 is an extremely thermostable recombinant protein from thermophilic archaeon Aeropyrumpernix K1. The Tyr444 located in the catalytic domain adjacent to the catalytic amino acid Ser445 a...Hyperthermophilic enzyme APE1547 is an extremely thermostable recombinant protein from thermophilic archaeon Aeropyrumpernix K1. The Tyr444 located in the catalytic domain adjacent to the catalytic amino acid Ser445 and formed hydrogen bond with Ile567. To study the effect of Tyr444 on the activity of APE1547, site-directed mutagenesis was applied. Two mutant enzymes T444S and T444G were created. Comparison of the mutant enzymes with wide enzyme, the thermostability of mutants T444S and T444G decreased by 10%-20%, but the catalytic efficiency of mutants toward pNPC8 and Ac-Leu-pNA increased 1.33 and 1.75 fold respectively. Molecular modeling shows that the elimination of hydrogen bond between Tyr444 and Ile567 is the cause of the decrease in thermostability and increase in catalytic efficiency. These observations suggest that Tyr444 plays an important role in the catalytic ability and thermostability of this enzyme.展开更多
The roles of nucleotide excision repair (NER) proteins in removing UV-induced lesions are well defined. There are two distinct NER pathways: global genome NER (GG-NER) and transcription-coupled NER. In human GG-NER, t...The roles of nucleotide excision repair (NER) proteins in removing UV-induced lesions are well defined. There are two distinct NER pathways: global genome NER (GG-NER) and transcription-coupled NER. In human GG-NER, two heteromeric protein complexes, DDB1-DDB2 and XPC-RAD23, are responsible for initial lesion recognition. Here, we examined the genetic interactions between GG-NER and base excision repair (BER) genes during abasic (AP) site repair of Schizosaccharomyces pombe. Mutants of rhp7 (rhp7-rhp16 are functional homologs of DDB1-DDB2) and rhp41 (XPC homolog) were moderately sensitive to methyl methanesulfonate and slightly to sodium bisulfite. Nth1p most actively cleaves the AP site in S. pombe. Deletion of rhp7 or rhp41 from nth1Δ cells greatly increased their sensitivity to alkylation and deamination, indicating that Rhp7p and Rhp41p are involved in repair of the AP sites generated by the action of DNA glycosylase. Induction of rhp7 and rhp16 genes by different types of DNA damage supports the ability of GG-NER to remove non-bulky lesions. Therefore, GG-NER activity not only targets bulky DNA helix-distorting lesions, but can also efficiently remove AP sites synergistically with BER.展开更多
目的研究尼古丁抑制DNA损伤后修复的分子机制。方法 Western blot检测蛋白表达水平,同位素方法检测Ref-1磷酸化和其核酸内切酶活性,试剂盒定量检测AP位点。结果尼古丁可以诱导Ref-1磷酸化,下调其核酸内切酶活性,抑制AP位点的修复,星状...目的研究尼古丁抑制DNA损伤后修复的分子机制。方法 Western blot检测蛋白表达水平,同位素方法检测Ref-1磷酸化和其核酸内切酶活性,试剂盒定量检测AP位点。结果尼古丁可以诱导Ref-1磷酸化,下调其核酸内切酶活性,抑制AP位点的修复,星状孢子素(Staurospo rine)和2-(2-氨基-3-甲氧基苯基)-4H-1-苯并吡喃-4-酮(PD98059)可以抑制尼古丁诱导的Ref-1磷酸化。结论尼古丁通过诱导Ref-1磷酸化,下调其核酸内切酶活性,抑制DNA损伤的修复。展开更多
Previous studies with deletion and sequence analysis of JDV LTR showed that there is a putative AP-4 responsive element in LTR. By antisense transient assay and gel shifting assay, we for the first time demonstrated t...Previous studies with deletion and sequence analysis of JDV LTR showed that there is a putative AP-4 responsive element in LTR. By antisense transient assay and gel shifting assay, we for the first time demonstrated that AP-4 modulated JDV gene expression by binding DNA di-rectly to bovine cells. The results, derived from site-directed mutagenesis experiments, suggest that the six base pairs of AP-4 binding site (CAGCTG) have different effects on JDV gene expression. When the first two base pairs changed to GC, JDV gene expression is severely decreased.展开更多
基金Supported by the National Natural Science Foundation of China(Nos.30400081 and 20432010)
文摘Hyperthermophilic enzyme APE1547 is an extremely thermostable recombinant protein from thermophilic archaeon Aeropyrumpernix K1. The Tyr444 located in the catalytic domain adjacent to the catalytic amino acid Ser445 and formed hydrogen bond with Ile567. To study the effect of Tyr444 on the activity of APE1547, site-directed mutagenesis was applied. Two mutant enzymes T444S and T444G were created. Comparison of the mutant enzymes with wide enzyme, the thermostability of mutants T444S and T444G decreased by 10%-20%, but the catalytic efficiency of mutants toward pNPC8 and Ac-Leu-pNA increased 1.33 and 1.75 fold respectively. Molecular modeling shows that the elimination of hydrogen bond between Tyr444 and Ile567 is the cause of the decrease in thermostability and increase in catalytic efficiency. These observations suggest that Tyr444 plays an important role in the catalytic ability and thermostability of this enzyme.
文摘The roles of nucleotide excision repair (NER) proteins in removing UV-induced lesions are well defined. There are two distinct NER pathways: global genome NER (GG-NER) and transcription-coupled NER. In human GG-NER, two heteromeric protein complexes, DDB1-DDB2 and XPC-RAD23, are responsible for initial lesion recognition. Here, we examined the genetic interactions between GG-NER and base excision repair (BER) genes during abasic (AP) site repair of Schizosaccharomyces pombe. Mutants of rhp7 (rhp7-rhp16 are functional homologs of DDB1-DDB2) and rhp41 (XPC homolog) were moderately sensitive to methyl methanesulfonate and slightly to sodium bisulfite. Nth1p most actively cleaves the AP site in S. pombe. Deletion of rhp7 or rhp41 from nth1Δ cells greatly increased their sensitivity to alkylation and deamination, indicating that Rhp7p and Rhp41p are involved in repair of the AP sites generated by the action of DNA glycosylase. Induction of rhp7 and rhp16 genes by different types of DNA damage supports the ability of GG-NER to remove non-bulky lesions. Therefore, GG-NER activity not only targets bulky DNA helix-distorting lesions, but can also efficiently remove AP sites synergistically with BER.
文摘目的研究尼古丁抑制DNA损伤后修复的分子机制。方法 Western blot检测蛋白表达水平,同位素方法检测Ref-1磷酸化和其核酸内切酶活性,试剂盒定量检测AP位点。结果尼古丁可以诱导Ref-1磷酸化,下调其核酸内切酶活性,抑制AP位点的修复,星状孢子素(Staurospo rine)和2-(2-氨基-3-甲氧基苯基)-4H-1-苯并吡喃-4-酮(PD98059)可以抑制尼古丁诱导的Ref-1磷酸化。结论尼古丁通过诱导Ref-1磷酸化,下调其核酸内切酶活性,抑制DNA损伤的修复。
文摘Previous studies with deletion and sequence analysis of JDV LTR showed that there is a putative AP-4 responsive element in LTR. By antisense transient assay and gel shifting assay, we for the first time demonstrated that AP-4 modulated JDV gene expression by binding DNA di-rectly to bovine cells. The results, derived from site-directed mutagenesis experiments, suggest that the six base pairs of AP-4 binding site (CAGCTG) have different effects on JDV gene expression. When the first two base pairs changed to GC, JDV gene expression is severely decreased.
