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

通过 UP1 蛋白恢复富含 G 重复序列中的 DNA 复制叉停滞

基本信息

批准号：
15570131
负责人：
FUKUDA Hirokazu
金额：
$ 2.37万
依托单位：
National Cancer Center (Research Institute)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

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.

在使用293细胞提取物的SV40无细胞DNA复制系统中，不能检测到模板质粒上的(CAGGG)n处的DNA复制停滞。可能是RECQ解旋酶家族中的一员UP1蛋白或提取物中的任何其他因素恢复了复制停滞。有必要使用这些因子缺乏或被击倒的细胞的提取物进行类似的实验。UP1基因敲除的HeLa细胞CGG重复数和染色体断裂数与对照细胞无明显差异。UP1或其他类UP1蛋白的泄漏表达可能补偿了UP1蛋白数量的减少。为了阐明另一种CAGGG重复序列结合蛋白hnRNP A3的功能，我们从大肠杆菌中纯化了重组蛋白，并对其与DNA结合的序列特异性进行了检测。HnRNP A3蛋白与d(CAGGG)n结合亲和力高，与端粒d(TTAGGG)n重复序列亲和力高。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UP1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细胞端粒长度的影响。