EFFICIENT SYSTEM OF HOMOLOGOUS RNA RECOMBINATION IN BROME MOSAIC-VIRUS - SEQUENCE AND STRUCTURE REQUIREMENTS AND ACCURACY OF CROSSOVERS
EFFICIENT SYSTEM OF HOMOLOGOUS RNA RECOMBINATION IN BROME MOSAIC-VIRUS - SEQUENCE AND STRUCTURE REQUIREMENTS AND ACCURACY OF CROSSOVERS
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DOI：
10.1128/jvi.69.1.131-140.1995
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发表时间：
1995-01-01
影响因子：
5.4
通讯作者：
BUJARSKI, JJ
中科院分区：
文献类型：
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作者：
NAGY, PD;BUJARSKI, JJ
溴花叶病毒（BMV）是一种支持片段间RNA重组的植物三边正链RNA病毒，用于确定同源杂交的序列和结构要求。野生型RNA2和突变型RNA3之间共有一个60个核苷酸（nt）的序列，通过与野生型RNA2 3‘非编码序列的同源重组，支持RNA3片段中修饰的3’非编码区域的有效修复（90%）。RNA3中该序列的缺失表明，短至15 nt的核苷酸身份可以支持高效的同源重组事件，而短至5 nt的序列身份导致该区域内重组频率降低（5%）。在常见的60-nt RNA3序列的下游部分，三个或三个以上的错配既影响了重组的发生率，也影响了交叉位点的分布，这表明除了长度外，两个重组BMV rna之间的序列一致性程度是同源重组的一个重要因素。对RNA3中共同序列的定点突变未发现预测二级结构的稳定性与重组活性之间存在明确的相关性。这表明同源重组不需要在交叉位点的两个重组rna之间有相似的二级结构。近20%的同源重组是不精确的（异常的），包含核苷酸错配，小缺失，或小插入在交叉区域。这意味着同源RNA重组并不像以前提出的那样准确。我们的研究结果提供了实验证据，证明BMV同源重组的要求和机制不同于先前描述的异源双工介导的非同源重组
Brome mosaic virus (BMV), a tripartite positive-stranded RNA virus of plants engineered to support intersegment RNA recombination, was used for the determination of sequence and structural requirements of homologous crossovers. A 60-nucleotide (nt) sequence, common between wild-type RNA2 and mutant RNA3, supported efficient repair (90%) of a modified 3' noncoding region in the RNA3 segment by homologous recombination with wild-type RNA2 3' noncoding sequences. Deletions within this sequence in RNA3 demonstrated that a nucleotide identity as short as 15 nt can support efficient homologous recombination events, while shorter (5-nt) sequence identity resulted in reduced recombination frequency (5%) within this region. Three or more mismatches within a downstream portion of the common 60-nt RNA3 sequence affected both the incidence of recombination and the distribution of crossover sites, suggesting that besides the length, the extent of sequence identity between two recombining BMV RNAs is ah important factor in homologous recombination. Site-directed mutagenesis of the common sequence in RNA3 did not reveal a clear correlation between the stability of predicted secondary structures and recombination activity. This indicates that homologous recombination does not require similar secondary structures between two recombining RNAs at the sites of crossovers. Nearly 20% of homologous recombinants were imprecise (aberrant), containing either nucleotide mismatches, small deletions, or small insertions within the region of crossovers. This implies that homologous RNA recombination is not as accurate as proposed previously. Our results provide experimental evidence that the requirements and thus the mechanism of homologous recombination in BMV differ from those of previously described heteroduplex-mediated nonhomologous recombination