The physical interaction of Mcm10 with Cdc45 modulates their DNA-binding properties

被引:17
作者
Di Perna, Roberta [1 ]
Aria, Valentina [1 ]
De Falco, Mariarosaria [1 ]
Sannino, Vincenzo [1 ]
Okorokov, Andrei L. [2 ]
Pisani, Francesca M. [1 ]
De Felice, Mariarita [1 ]
机构
[1] CNR, Ist Biochim Prot, I-80131 Naples, Italy
[2] UCL, Wolfson Inst Biomed Res, London WC1E 6BT, England
基金
英国医学研究理事会;
关键词
Cdc45; cell cycle; DNA replication; genome stability; Mcm10; POLYMERASE-ALPHA; REPLICATION ORIGINS; CATALYTIC SUBUNIT; INITIATION; CHROMATIN; HELICASE; DOMAIN; ASSOCIATION; COMPLEX; MCM2-7;
D O I
10.1042/BJ20130059
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The eukaryotic DNA replication protein Mcm10 (mini-chromosome maintenance 10) associates with chromatin in early S-phase and is required for assembly and function of the replication fork protein machinery. Another essential component of the eukaryotic replication fork is Cdc45 (cell division cycle 45), which is required for both initiation and elongation of DNA replication. In the present study we characterize, for the first time, the physical and functional interactions of human Mcm10 and Cdc45. First we demonstrated that Mcm10 and Cdc45 interact in cell-free extracts. We then analysed the role of each of the Mcm10 domains: N-terminal, internal and C-terminal (NTD, ID and CTD respectively). We have detected a direct physical interaction between CTD and Cdc45 by both in vitro co-immunoprecipitation and surface plasmon resonance experiments. On the other hand, we have found that the interaction of the Mcm10 ID with Cdc45 takes place only in the presence of DNA. Furthermore, we found that the isolated ID and CTD domains are fully functional, retaining DNA-binding capability with a clear preference for bubble and fork structures, and that they both enhance Cdc45 DNA-binding affinity. The results of the present study demonstrate that human Mcm10 and Cdc45 directly interact and establish a mutual co-operation in DNA binding.
引用
收藏
页码:333 / 343
页数:11
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