Yu K, Lieber M R
Norris Comprehensive Cancer Center and Departments of Pathology, University of Southern California School of Medicine, Los Angeles, CA 90089-9176, USA.
Mol Cell Biol. 2000 Nov;20(21):7914-21. doi: 10.1128/MCB.20.21.7914-7921.2000.
In all of the transposition reactions that have been characterized thus far, synapsis of two transposon ends is required before any catalytic steps (strand nicking or strand transfer) occur. In V(D)J recombination, there have been inconclusive data concerning the role of synapsis in nicking. Synapsis between two 12-substrates or between two 23-substrates has not been ruled out in any studies thus far. Here we provide the first direct tests of this issue. We find that immobilization of signals does not affect their nicking, even though hairpinning is affected in a manner reflecting its known synaptic requirement. We also find that nicking is kinetically a unireactant enzyme-catalyzed reaction. Time courses are no different between nicking seen for a 12-substrate alone and a reaction involving both a 12- and a 23-substrate. Hence, synapsis is neither a requirement nor an effector of the rate of nicking. These results establish V(D)J recombination as the first example of a DNA transposition-type reaction in which catalytic steps begin prior to synapsis, and the results have direct implications for the order of the steps in V(D)J recombination, for the contribution of V(D)J recombination nicks to genomic instability, and for the diversification of the immune repertoire.
在迄今为止已被表征的所有转座反应中，在任何催化步骤（链切割或链转移）发生之前，都需要两个转座子末端进行联会。在V(D)J重组中，关于联会在切割中所起作用的数据尚无定论。到目前为止，在任何研究中都没有排除两个12碱基底物之间或两个23碱基底物之间的联会。在此，我们首次对这个问题进行了直接测试。我们发现，信号的固定化并不影响它们的切割，尽管发夹结构的形成受到影响，其方式反映了其已知的联会需求。我们还发现，切割在动力学上是一个单反应物酶催化反应。单独一个12碱基底物的切割与一个同时涉及12碱基和23碱基底物的反应之间的时间进程并无差异。因此，联会既不是切割速率的必要条件，也不是其影响因素。这些结果确立了V(D)J重组作为DNA转座型反应的首个例子，其中催化步骤在联会之前就开始了，并且这些结果对于V(D)J重组步骤的顺序、V(D)J重组切割对基因组不稳定性的影响以及免疫库的多样化具有直接意义。
