Costanzo Vincenzo, Shechter David, Lupardus Patrick J, Cimprich Karlene A, Gottesman Max, Gautier Jean
Department of Genetics and Development, Columbia University, New York, NY 10032, USA.
Mol Cell. 2003 Jan;11(1):203-13. doi: 10.1016/s1097-2765(02)00799-2.
We have analyzed how single-strand DNA gaps affect DNA replication in Xenopus egg extracts. DNA lesions generated by etoposide, a DNA topoisomerase II inhibitor, or by exonuclease treatment activate a DNA damage checkpoint that blocks initiation of plasmid and chromosomal DNA replication. The checkpoint is abrogated by caffeine and requires ATR, but not ATM, protein kinase. The block to DNA synthesis is due to inhibition of Cdc7/Dbf4 protein kinase activity and the subsequent failure of Cdc45 to bind to chromatin. The checkpoint does not require pre-RC assembly but requires loading of the single-strand binding protein, RPA, on chromatin. This is the biochemical demonstration of a DNA damage checkpoint that targets Cdc7/Dbf4 protein kinase.
我们分析了单链DNA缺口如何影响非洲爪蟾卵提取物中的DNA复制。由拓扑异构酶II抑制剂依托泊苷或核酸外切酶处理产生的DNA损伤会激活DNA损伤检查点，该检查点会阻断质粒和染色体DNA复制的起始。咖啡因可消除该检查点，且该检查点需要ATR蛋白激酶，但不需要ATM蛋白激酶。DNA合成的阻断是由于Cdc7/Dbf4蛋白激酶活性受到抑制，以及随后Cdc45无法与染色质结合。该检查点不需要前复制复合体组装，但需要单链结合蛋白RPA加载到染色质上。这是针对Cdc7/Dbf4蛋白激酶的DNA损伤检查点的生化证明。
