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V(D)J recombination: evidence that a replicative mechanism is not required.

机译：V（D）J重组：不需要复制机制的证据。

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We examined a series of extrachromosomal DNA substrates for V(D)J recombination under replicating and nonreplicating conditions. Complete and partial replications were examined by monitoring the loss of prokaryote-specific adenine methylation at 14 to 22 MboI-DpnI restriction sites (GATC) on the substrates. Some of these sites are within 2 bases of the signal sequence ends. We found that neither coding joint nor signal joint formation requires substrate replication. After ruling out replication as a substrate requirement, we determined whether replication had any effect on the efficiency of V(D)J recombination. Quantitation of V(D)J recombination efficiency on nonreplicating substrates requires some method of monitoring the entry of substrate molecules into the cells. We devised such a method by monitoring DNA repair of substrates into which we had substituted deoxyuridine for 10 to 20% of the thymidine nucleotides in the DNA. The substrates which enter the lymphoid cells were repaired efficiently in vivo by the eukaryotic uracil DNA repair system. Upon plasmid harvest, we distinguished repaired (entered) from unrepaired (not entered) plasmids by cleaving unrepaired molecules with uracil DNA glycoylase and Escherichia coli endonuclease IV in vitro. This method of monitoring DNA entry does not appear to underestimate or overestimate the amount of DNA entry. By using this method, we found no significant quantitative effect of DNA replication on V(D)J recombination efficiency.
机译：我们检查了一系列在复制和非复制条件下V（D）J重组的染色体外DNA底物。通过监测底物上14至22个MboI-DpnI限制性位点（GATC）上原核生物特异性腺嘌呤甲基化的丧失来检查完整和部分复制。这些位点中的一些在信号序列末端的2个碱基内。我们发现编码接头或信号接头的形成都不需要底物复制。在排除将复制作为底物要求之后，我们确定复制是否对V（D）J重组效率有任何影响。定量非复制底物上的V（D）J重组效率需要一些监测底物分子进入细胞的方法。我们通过监测底物的DNA修复来设计这种方法，在该底物中我们用脱氧尿苷取代了DNA中10至20％的胸苷核苷酸。进入淋巴样细胞的底物在体内被真核尿嘧啶DNA修复系统有效地修复。收获质粒后，我们通过在体外用尿嘧啶DNA糖基化酶和大肠杆菌内切核酸酶IV切割未修复的分子，将未修复（未进入）的质粒与修复的（输入的）质粒区分开。这种监视DNA进入的方法似乎并未低估或高估了DNA进入的数量。通过使用这种方法，我们发现DNA复制对V（D）J重组效率没有明显的定量影响。

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