MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens.
MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens.
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DOI：
10.1093/nar/gkr1272
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发表时间：
2012-04
影响因子：
14.9
通讯作者：
Nogué F
中科院分区：
文献类型：
--
作者：
Kamisugi Y;Schaefer DG;Kozak J;Charlot F;Vrielynck N;Holá M;Angelis KJ;Cuming AC;Nogué F
藓类小立碗藓在植物模型中是独特的，因为通过同源重组发生靶向转基因插入的频率很高。转基因整合被认为是利用现有的检测和修复DNA双链断裂（DSB）的机制。我们进行了有针对性的敲除立碗藓基因编码的主要传感器的DNA DSB，MRN复合物的组件。PpMRE 11或PpRAD 50的功能丧失强烈且特异性地抑制基因靶向，而非靶向转基因整合的速率相对不受影响。相反，破坏的PpNBS 1基因保留了野生型的能力，有效地整合转化DNA在同源位点。通过单核琼脂糖凝胶电泳分析野生型和突变体植物中DNA-DSB修复的动力学发现，博莱霉素诱导的基因组DNA片段在每个基因型中以大致相等的速率修复，尽管Ppmre 11和Pprad 50突变体与野生型和Ppnbs 1植物相比，表现出严重限制的生长和发育，并增强了对UV-B和博莱霉素诱导的DNA损伤的敏感性。这意味着，虽然广泛的DNA修复可以在没有功能性MRN复合物的情况下发生;但这在本质上是不受监督的，并导致与正常生长和发育不相容的有害突变的积累。
The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development.
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影响因子：
16
作者：
Deriano, Ludovic;Stracker, Travis H.;Baker, Annalee;Petrini, John H. J.;Roth, David B.
通讯作者：
Roth, David B.
影响因子：
4.8
作者：
Della-Maria, Julie;Zhou, Yi;Tomkinson, Alan E.
通讯作者：
Tomkinson, Alan E.
影响因子：
11.4
作者：
Heacock, M;Spangler, E;Shippen, DE
通讯作者：
Shippen, DE
影响因子：
11.6
作者：
Bundock, P;Hooykaas, P
通讯作者：
Hooykaas, P
影响因子：
5.1
作者：
Kamisugi, Y;Cuming, AC
通讯作者：
Cuming, AC