Restoring DNA replication fork arrest within G-rich repetitive sequences by UP1 protein

在使用293细胞提取物的SV 40无细胞DNA复制系统中，在模板质粒上的（CAGGG）n处不能检测到DNA复制停滞。这可能是UP 1蛋白，RecQ解旋酶家族蛋白的成员，或提取物中的任何其他因子恢复复制停滞。有必要使用这些因子缺乏或被敲除的细胞提取物进行类似的实验。UP 1基因敲低的HeLa细胞CGG重复数和染色体断裂数与对照组无明显差异。UP 1或其他UP 1样蛋白的漏表达可能补偿了UP 1蛋白量的减少。为了阐明hnRNPA 3蛋白的功能，我们从大肠杆菌中纯化了一个重组蛋白，并检测了其DNA结合的序列特异性。hnRNP A3蛋白以高亲和力结合d（CAGGG）n，并且以高得多的亲和力结合端粒d（TTAGGG）n重复。在使用纯化的DNA聚合酶和携带（CAGGG）n或（TTAGGG）n重复序列的单链DNA模板的体外DNA复制系统中，hnRNP A3蛋白对重复序列内的DNA合成具有抑制作用，与UP 1相反。hnRNP A3蛋白在体外被证明可以保护TTAGGG重复序列免受核酸酶的攻击。我们现在正在阐明hnRNP A3过表达对tet-on HeLa细胞中端粒长度的影响。

DNA replication arrest at (CAGGG)n on a template plasmid could not been detected in an SV40 cell-free DNA replication system using extracts from 293 cells. It is possible that UP1 protein, a member of RecQ helicase family proteins, or any other factors in the extracts restored the replication arrest. It will be necessary to carry out similar experiments using extracts from the cells in which these factors are deficient or knocked down. There was no difference of the number of CGG repeat and chromosomal breaks between UP1-knocked-down HeLa cells and control cells. It is possible that leaky expression of UP1 or other UP1-like proteins compensated for the decrease in the amount of UP1 proteins. In order to clarify the functions of the hnRNP A3 protein, which was identified as another CAGGG repeat binding protein, we purified a recombinant protein from E.coli and examined the sequence-specificity of its DNA-binding. hnRNP A3 protein bound to d(CAGGG)n with a high affinity, and to telomeric d(TTAGGG)n repeats with a much higher affinity. In an in vitro DNA replication system using purified DNA polymerase and single-stranded DNA templates carrying (CAGGG)n or (TTAGGG)n repeats, hnRNP A3 proteins had an inhibitory effect on DNA synthesis within the repeats, contrary to UP1. hnRNP A3 protein was demonstrated in vitro to protect TTAGGG repeat from an attack of nucleases. We are now elucidating the effect of over-expression of hnRNP A3 on telomere-length in tet-on HeLa cells.