Department of Genetics, Iowa State University, Ames, IA 50011, USA.
Nucleic Acids Res. 2011 May;39(10):4166-79. doi: 10.1093/nar/gkq1363. Epub 2011 Jan 25.
The bacterial recombinase RecA forms a nucleic acid-protein filament on single-stranded (ss) DNA during the repair of double-strand breaks (DSBs) that efficiently undergoes a homology search and engages in pairing with the complementary DNA sequence. We utilized the pairing activity of RecA-DNA filaments to tether biochemical activities to specific chromosomal sites. Different filaments with chimeric RecA proteins were tested for the ability to induce loss of heterozygosity at the golden locus in zebrafish after injection at the one-cell stage. A fusion protein between RecA containing a nuclear localization signal (NLS) and the DNA-binding domain of Gal4 (NLS-RecA-Gal4) displayed the most activity. Our results demonstrate that complementary ssDNA filaments as short as 60 nucleotides coated with NLS-RecA-Gal4 protein are able to cause loss of heterozygosity in ∼3% of the injected embryos. We demonstrate that lesions in ∼9% of the F0 zebrafish are transmitted to subsequent generations as large chromosomal deletions. Co-injection of linear DNA with the NLS-RecA-Gal4 DNA filaments promotes the insertion of the DNA into targeted genomic locations. Our data support a model whereby NLS-RecA-Gal4 DNA filaments bind to complementary target sites on chromatin and stall DNA replication forks, resulting in a DNA DSB.
在修复双链断裂 (DSB) 时，细菌重组酶 RecA 会在单链 DNA 上形成核酸-蛋白质丝，该丝能够有效地进行同源搜索并与互补的 DNA 序列配对。我们利用 RecA-DNA 丝的配对活性将生化活性固定在特定的染色体位点上。我们测试了不同带有嵌合 RecA 蛋白的丝在注射到斑马鱼的单细胞期后，是否有能力在金色基因座诱导杂合性丢失。一种含有核定位信号 (NLS) 和 Gal4 DNA 结合域的 RecA 融合蛋白 (NLS-RecA-Gal4) 显示出最强的活性。我们的结果表明，带有 NLS-RecA-Gal4 蛋白的短至 60 个核苷酸的互补 ssDNA 丝能够导致约 3%的注射胚胎发生杂合性丢失。我们证明，约 9%的 F0 斑马鱼中的损伤会作为大的染色体缺失传递给后代。NLS-RecA-Gal4 DNA 丝与线性 DNA 的共注射促进了 DNA 插入到靶向基因组位置。我们的数据支持这样一种模型，即 NLS-RecA-Gal4 DNA 丝结合到染色质上的互补靶位点并使 DNA 复制叉停滞，从而导致 DNA DSB。
