RELAXASE (TRAI) OF INCP-ALPHA PLASMID RP4 CATALYZES A SITE-SPECIFIC CLEAVING-JOINING REACTION OF SINGLE-STRANDED-DNA
RELAXASE (TRAI) OF INCP-ALPHA PLASMID RP4 CATALYZES A SITE-SPECIFIC CLEAVING-JOINING REACTION OF SINGLE-STRANDED-DNA
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DOI：
10.1073/pnas.90.7.2925
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发表时间：
1993-04-01
影响因子：
11.1
通讯作者：
LANKA, E
中科院分区：
文献类型：
--
作者：
PANSEGRAU, W;SCHRODER, W;LANKA, E
自身传播的广宿主范围质粒RP 4的接合DNA转移通过在转移起点（oriT）的切口位点（nic）处的链特异性和位点特异性切割来启动。裂解导致质粒编码的松弛酶（TraI）共价连接到5 '-末端2'-脱氧胞苷残基的nic。我们证明，Tyr 22是TraI的催化位点的中心，介导通过形成DNA 5'磷酰基和芳香族羟基之间的磷酸二酯的裂解。用包含切口区域的短寡脱氧核苷酸验证了I型oriT DNA切割的特异性。该反应需要Tral和Mg 2+，但不依赖于松弛体组分TraJ。裂解产生一个具有游离3'羟基的寡核苷酸片段，另一部分形成共价的Tral-寡核苷酸加合物。与I型oriT DNA的切口一样，TraI催化的寡核苷酸切割在约30%的输入TraI作为共价蛋白质-DNA复合物存在时达到平衡。在两种不同大小的寡核苷酸的存在下，产生确定的杂合寡核苷酸，表明Tral催化两条单链在nic处的重组。这一发现表明，Tral具有类似于I型拓扑异构酶的切割-连接活性。反应依赖于与nic相邻的3 '端6个核苷酸的序列。只有少数位置的某些碱基变化是容许的，而5'末端核苷酸的序列显然与TraI的识别无关。本文所述的反应进一步支持了这样的假设，即通过缀合的DNA转移涉及滚环样机制，该机制产生移民单链，而DNA结合的Tral蛋白扫描供体-受体界面处第二切割位点的发生。
Conjugative DNA transfer of the self-transmissible broad-host-range plasmid RP4 is initiated by strand- and site-specific cleavage at the nick site (nic) of the transfer origin (oriT). Cleavage results in covalent attachment of the plasmid-encoded relaxase (TraI) to the 5'-terminal 2'-deoxycytidine residue at nic. We demonstrate that Tyr22 is the center of the catalytic site of TraI, mediating cleavage via formation of a phosphodiester between the DNA 5' phosphoryl and the aromatic hydroxyl group. The specificity of cleavage seen with form I oriT DNA was verified with short oligodeoxyribonucleotides embracing the nick region. The reaction requires Tral and Mg2+ but is independent of the relaxosome component TraJ. Cleavage produces one oligonucleotide fragment with a free 3' hydroxyl, the other part forms a covalent Tral-oligonucleotide adduct. Like nicking of form I oriT DNA, TraI-catalyzed oligonucleotide cleavage reaches an equilibrium when about 30% of the input Tral exists as a covalent protein-DNA complex. In the presence of two differently sized oligonucleotides, defined hybrid oligonucleotides are produced, demonstrating that Tral catalyzes recombination of two single strands at nic. This finding shows that Tral possesses cleaving-joining activity resembling that of a type I topoisomerase. Reactions are dependent on the sequence of the 3'-terminal 6 nucleotides adjacent to nic. Only certain base changes in a few positions are tolerated, whereas the sequence of the 5' terminal nucleotides apparently is irrelevant for recognition by TraI. The reactions described here further support the hypothesis that DNA transfer via conjugation involves a rolling circle-like mechanism which generates the immigrant single strand while DNA-bound Tral protein scans for the occurrence of a second cleavage site at the donor-recipient interface.