Brown M L, Chang Y
Department of Microbiology, Arizona State University, Tempe, AZ 85287, USA.
J Immunol. 2000 Apr 15;164(8):4135-42. doi: 10.4049/jimmunol.164.8.4135.
V(D)J recombination cleavage generates two types of dsDNA breaks: blunt signal ends and covalently sealed hairpin coding ends. Although signal ends can be directly ligated to form signal joints, hairpin coding ends need to be opened and subsequently processed before being joined. However, the underlying mechanism of coding end resolution remains undefined. The current study attempts to delineate this process by analyzing various structures of coding ends made in situ from recombination-inducible pre-B cell lines of both normal and scid mice. These cell lines were derived by transformation of B cell precursors with the temperature-sensitive Abelson murine leukemia virus. Our kinetic analysis revealed that under conditions permissive to scid transformants, hairpin coding ends could be nicked to generate 3' overhangs and then processed into blunt ends. The final joining of these blunt ends followed the same kinetics as signal joint formation. The course of this process is in sharp contrast to coding end resolution in scid heterozygous transformants that express the catalytic subunit of DNA-dependent protein kinase, in which hairpin end opening, processing, and joining proceeded very rapidly and appeared to be closely linked. Furthermore, we demonstrated that the opening of hairpin ends in scid cells could be manipulated by different culture conditions, which ultimately influenced not only the level and integrity of the newly formed coding joints, but also the extent of microhomology at the coding junctions. These results are discussed in the context of scid leaky recombination.
V(D)J重组切割产生两种双链DNA断裂：平端信号末端和共价封闭的发夹编码末端。虽然信号末端可直接连接形成信号接头，但发夹编码末端在连接之前需要打开并随后进行加工。然而，编码末端解析的潜在机制仍不明确。当前的研究试图通过分析从正常和scid小鼠的重组诱导前B细胞系原位产生的编码末端的各种结构来描绘这一过程。这些细胞系是通过用温度敏感型Abelson小鼠白血病病毒转化B细胞前体而获得的。我们的动力学分析表明，在允许scid转化体生长的条件下，发夹编码末端可被切口以产生3'端突出，然后加工成平端。这些平端的最终连接遵循与信号接头形成相同的动力学。这一过程与表达DNA依赖性蛋白激酶催化亚基的scid杂合转化体中的编码末端解析形成鲜明对比，在scid杂合转化体中，发夹末端的打开、加工和连接进行得非常迅速，并且似乎紧密相连。此外，我们证明scid细胞中发夹末端的打开可通过不同的培养条件进行调控，这最终不仅影响新形成的编码接头的水平和完整性，还影响编码连接处的微同源程度。这些结果将在scid渗漏重组的背景下进行讨论。
