Efficient concerted integration of retrovirus-like DNA in vitro by avian myeloblastosis virus integrase.
Efficient concerted integration of retrovirus-like DNA in vitro by avian myeloblastosis virus integrase.
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禽成髓细胞瘤病毒整合酶在体外对逆转录病毒样 DNA 进行高效协同整合。
DOI：
10.1093/nar/22.21.4454
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发表时间：
1994
影响因子：
14.9
通讯作者：
Grandgenett,DP
中科院分区：
文献类型：
--
作者：
Vora,AC;McCord,M;Fitzgerald,ML;Inman,RB;Grandgenett,DP
我们报告了从禽成髓细胞瘤病毒中纯化的整合酶（IN）将线性病毒样DNA供体有效地协同整合到2.8 kbp的环状DNA靶中。供体为528 bp，含有凹陷的3′ OH末端，5′末端标记，并且具有在靶中未发现的独特限制性位点。通过限制性内切酶分析和琼脂糖凝胶电泳完成协同（全位点）和半位点整合事件的分析。供体还含有SupF基因，其用于个体全位点重组体的遗传选择以确定宿主重复大小。两种不同的途径，涉及一个供体或两个供体分子，用于产生全位点重组体。约90%的全位点重组体是每个靶标使用两个供体分子的结果。这些结果意味着通过IN将来自两个供体中的每一个的末端并置比将单个供体的两个末端并置用于全位点反应更有效。在加入靶之前，在冰上形成含有整合酶和供体的整合前复合物增强了全位点反应。在37°C下反应30分钟后，所有供体/靶重组体的约20 - 25%是协同整合事件的结果。全位点重组子的高效生产需要Mg 2 + Mn 2+，仅对半位点重组子的生产有效。我们认为，这些预整合复合物可以用来研究3′ OH修剪和链转移反应之间的关系。
We report the efficient concerted integration of a linear virus-like DNA donor into a 2.8 kbp circular DNA target by integrase (IN) purified from avian myeloblastosis virus. The donor was 528 bp, contained recessed 3′ OH ends, was 5′ end labeled, and had a unique restriction site not found in the target. Analysis of concerted (fullsite) and half-site integration events was accomplished by restriction enzyme analysis and agarose gel electrophoresis. The donor also contained the SupF gene that was used for genetic selection of individual full-site recombinants to determine the host duplication size. Two different pathways, involving either one donor or two donor molecules, were used to produce full-site recombinants. About 90% of the full-site recombinants were the result of using two donor molecules per target. These results imply that juxtapositioning an end from each of two donors by IN was more efficient than the juxtapositioning of two ends of a single donor for the full-site reaction. The formation of preintegration complexes containing integrase and donor on ice prior to the addition of target enhanced the full-site reaction. After a 30 min reaction at 37°C, –20–25% of all donor/target recombinants were the result of concerted integration events. The efficient production of full-site recombinants required Mg2+Mn2+was only efficient for the production of half-site recombinants. We suggest that these preintegration complexes can be used to investigate the relationships between the 3′ OH trimming and strand transfer reactions.