An intrastrand three-DNA-base interaction is a key specificity determinant of F transfer initiation and of F TraI relaxase DNA recognition and cleavage.
An intrastrand three-DNA-base interaction is a key specificity determinant of F transfer initiation and of F TraI relaxase DNA recognition and cleavage.
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DOI：
10.1093/nar/gkn422
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发表时间：
2008-08
影响因子：
14.9
通讯作者：
Schildbach JF
中科院分区：
文献类型：
--
作者：
Hekman K;Guja K;Larkin C;Schildbach JF
细菌接合，在细菌之间转移单个接合质粒链，使原核基因组多样化并传播抗生素抗性基因。作为转移的先决条件，质粒编码的松弛酶以序列特异性结合并切割转移的质粒链。F TraI松弛酶结构域与结合单链DNA的晶体结构表明结合特异性部分由链内三向碱基配对相互作用决定。我们以前表明，三个相互作用的碱基的单一取代可以显着降低结合。在这里，我们研究了单和双碱基取代在这些位置上的质粒动员的效果。许多取代减少了转移，尽管一些取代的有害影响可以通过在第二位点的取代而部分克服。我们测量了F TraI松弛酶结构域对几种DNA序列变体的亲和力。虽然降低的转移通常与降低的结合亲和力相关，但一些oriT变体的转移效率与其结合亲和力的预期不同，表明ssDNA结合和切割并不绝对相关。寡核苷酸切割试验结果表明，三碱基相互作用的基本功能可能是定位易切割的磷酸盐进行切割，而不是直接有助于结合亲和力。
Bacterial conjugation, transfer of a single conjugative plasmid strand between bacteria, diversifies prokaryotic genomes and disseminates antibiotic resistance genes. As a prerequisite for transfer, plasmid-encoded relaxases bind to and cleave the transferred plasmid strand with sequence specificity. The crystal structure of the F TraI relaxase domain with bound single-stranded DNA suggests binding specificity is partly determined by an intrastrand three-way base-pairing interaction. We showed previously that single substitutions for the three interacting bases could significantly reduce binding. Here we examine the effect of single and double base substitutions at these positions on plasmid mobilization. Many substitutions reduce transfer, although the detrimental effects of some substitutions can be partially overcome by substitutions at a second site. We measured the affinity of the F TraI relaxase domain for several DNA sequence variants. While reduced transfer generally correlates with reduced binding affinity, some oriT variants transfer with an efficiency different than expected from their binding affinities, indicating ssDNA binding and cleavage do not correlate absolutely. Oligonucleotide cleavage assay results suggest the essential function of the three-base interaction may be to position the scissile phosphate for cleavage, rather than to directly contribute to binding affinity.
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影响因子：
5.6
作者：
Boer, R;Russi, S;de la Cruz, F
通讯作者：
de la Cruz, F
影响因子：
3.2
作者：
Fekete, RA;Frost, LS
通讯作者：
Frost, LS
影响因子：
3.1
作者：
Hill, VT;Townsend, SM;Badger, JL
通讯作者：
Badger, JL
影响因子：
14.9
作者：
Williams, SL;Schildbach, JF
通讯作者：
Schildbach, JF
DOI：
10.1073/pnas.0506081102
发表时间：
2005-11-08
影响因子：
11.1
作者：
Draper, O;César, CE;Llosa, M
通讯作者：
Llosa, M