Eastman Q M, Schatz D G
Department of Molecular Biophysics and Biochemistry and Section of Immunobiology, Yale University School of Medicine, Howard Hughes Medical Institute, New Haven, CT 06520-8011, USA.
Nucleic Acids Res. 1997 Nov 1;25(21):4370-8. doi: 10.1093/nar/25.21.4370.
The first step in DNA cleavage at V(D)J recombination signals by RAG1 and RAG2 is creation of a nick at the heptamer/coding flank border. Under proper conditions in vitro the second step, hairpin formation, requires two signals with spacers of 12 and 23 bp, a restriction referred to as the 12/23 rule. Under these conditions hairpin formation occurs at the two signals at or near the same time. In contrast, we find that under the same conditions nicking occurs at isolated signals and hence is not subject to the 12/23 rule. With two signals the nicking events are not concerted and the signal with a 12 bp spacer is usually nicked first. However, the extent and rate of nicking at a given signal are diminished by mutations of the other signal. The appearance of DNA nicked at both signals is stimulated by more than an order of magnitude by the ability of the signals to synapse, indicating that synapsis accelerates nicking and often precedes it. These observations allow formulation of a more complete model of catalysis of DNA cleavage and how the 12/23 rule is enforced.
RAG1和RAG2在V(D)J重排信号处切割DNA的第一步是在七聚体/编码侧翼边界处产生一个切口。在体外适当条件下，第二步即发夹形成，需要两个间隔为12和23个碱基对的信号，这一限制被称为12/23规则。在这些条件下，发夹形成在两个信号处或几乎同时发生。相比之下，我们发现，在相同条件下，切口在孤立信号处产生，因此不受12/23规则的限制。对于两个信号，切口事件并非协同发生，具有12个碱基对间隔的信号通常首先被切开。然而，给定信号处的切口程度和速率会因另一个信号的突变而降低。信号进行突触连接的能力使两个信号处都出现切口的情况被刺激了一个数量级以上，这表明突触连接加速了切口形成，并且通常先于切口形成。这些观察结果有助于构建一个更完整的DNA切割催化模型以及12/23规则是如何被执行的。
