权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Cleavage and Rejoining of DNA in the Shufflon-Specific Recombinase

Shufflon 特异性重组酶中 DNA 的切割和重新连接

基本信息

批准号：
10216207
负责人：
KOMANO Teruya
金额：
$ 24.06万
依托单位：
Tokyo Metropolitan University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10216207/
关键词：
shufflon site-specific recombination plasmid R64 conjugal transfer cleavage and rejoining of DNA

项目摘要

The pilV gene, the last gene of the pil operon of plasmid R64 is under the control of shufflon multiple inversion. R64 shufflon consists of four DNA segments, separated and flanked by seven sfx sequences. Site-specific recombinations mediated by the rci product occur between any inverted, sfx sequences, resulting. in inversions of the four segments independently or in groups. Thus the C-terminal segments of PilV adhesins interconvert among seven types, which determine the recipient specificity in R64 liquid matings.Rci protein was overproduced and purified. Specific cleavages were introduced into the sfx sequence when a sfx DNA fragment was incubated with Rci. Cleavages occurred in the form of a 5' protruding 7-bp staggered cut, suggesting that DNA cleavage and rejoining in the shufflon recombination take place at these positions. The sfx sequences were shown to consist of central 7-bp spacer sequence and left and right 12-bp arms, where the sfx left arm sequence are not conserved. Rci tein was shown to bind to entire sfx sequences, suggesting that one Rci molecule binds to the conserved sfx right arm in a sequence-specific manner and the second Rci molecule binds to the sfx left arm in a non-sequence-specific manner. Different sfx left arm sequences exhibited different inversion frequencies. When a symmetric sfx sequence, where the left and right arm sequences are the same, was constructed, recombination between symmetric sfx sequences in direct orientation was observed. Asymmetry of the sfx sequence may be the reason why Rci protein acts on only the inverted sfx sequences. R64 liquid matings using various waa mutants of E. coli K-12 and S. enterica serovar Typhimurium LT2 as recipient strains indicated that the specific receptors for the PilV adhesins are LPS molecules. Binding of PilV adhesins to respective LPS molecules was demonstrated.

pilV基因是质粒R64的操纵子的最后一个基因，受shufflon多重倒位控制。R64 shufflon由四个DNA片段组成，由七个sfx序列分隔和侧翼。由rci产物介导的位点特异性重组发生在任何反向的sfx序列之间。在四个部分的倒置中独立地或成组地。因此，PilV粘附素的C端片段在7种类型之间相互转换，决定了R64液体交配中的受体特异性。当sfx DNA片段与Rci孵育时，将特异性切割引入sfx序列。切割以5'突出的7-bp交错切割的形式发生，表明在shufflon重组中的DNA切割和重新连接发生在这些位置。sfx序列显示由中央7-bp间隔序列和左右12-bp臂组成，其中sfx左臂序列不保守。显示Rci蛋白结合整个sfx序列，表明一个Rci分子以序列特异性方式结合保守的sfx右臂，第二个Rci分子以非序列特异性方式结合sfx左臂。不同的sfx左臂序列表现出不同的倒位频率。当构建对称sfx序列时，其中左臂和右臂序列相同，观察到对称sfx序列之间以直接取向的重组。sfx序列的不对称性可能是Rci蛋白仅作用于反向sfx序列的原因。R64液体交配，利用各种waa突变体的E。coli K-12和S.肠道血清型鼠伤寒LT 2作为受体菌株表明PilV粘附素的特异性受体是LPS分子。证明了PilV粘附素与相应LPS分子的结合。