Cleavage and Rejoining of DNA in the Shufflon-Specific Recombinase

PilV基因是R64载体pIL操纵子的最后一个基因，受Shufflon多重倒位控制。R64Shufflon由四个DNA片段组成，由七个SFX序列隔开并位于其两侧。由RCI产物介导的位点特异性重组发生在任何倒置的SFX序列之间，结果是。在四个分段的倒置中独立地或成组地。因此，PilV粘附素的C末端片段在7种类型之间相互转换，这决定了R64液体配对的受体特异性。Rci蛋白得到了过量生产和纯化。当SFX DNA片段与RCI孵育时，SFX序列中引入了特定的切割。裂解以5‘突起的7-碱基交错切开的形式出现，这表明DNA裂解和重新加入改组重组发生在这些位置。SFX序列由中央7个碱基的间隔区序列和左右12个碱基的臂组成，其中SFX的左臂序列不保守。RCI tein被证明与整个SFX序列结合，这表明一个RCI分子以序列特异性的方式与保守的SFX右臂结合，第二个RCI分子以非序列特异性的方式与SFX左臂结合。不同的SFX左臂序列表现出不同的倒置频率。当构建左臂和右臂序列相同的对称SFX序列时，观察到对称SFX序列之间的直接定向重组。SFX序列的不对称性可能是RCI蛋白只作用于反向SFX序列的原因。用大肠杆菌K-12的各种waa突变体和鼠伤寒沙门氏菌LT2的不同waa突变体作为受体菌株进行的R64液体配对表明，PilV粘附素的特异性受体是内毒素分子。PilV粘附素可与相应的内毒素分子结合。

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.