Dhingra Nalini, Bruck Irina, Smith Skye, Ning Boting, Kaplan Daniel L
From the Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235 and.
the Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida 32306-4300.
J Biol Chem. 2015 Mar 20;290(12):7586-601. doi: 10.1074/jbc.M115.640383. Epub 2015 Feb 6.
Dpb11 is required for the initiation of DNA replication in budding yeast. Dpb11 binds to S-phase cyclin-dependent kinase-phosphorylated Sld2 and Sld3 to form a ternary complex during S phase. The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7, and GINS. We show here, using purified proteins from budding yeast, that Dpb11 alone binds to Mcm2-7 and that Dpb11 also competes with GINS for binding to Mcm2-7. Furthermore, Dpb11 binds directly to single-stranded DNA (ssDNA), and ssDNA inhibits the Dpb11 interaction with Mcm2-7. We also found that Dpb11 can recruit Cdc45 to Mcm2-7. We identified a mutant of the BRCT4 motif of Dpb11 that remains bound to Mcm2-7 in the presence of ssDNA (dpb11-m1,m2,m3,m5), and this mutant exhibits a DNA replication defect when expressed in budding yeast cells. Expression of this mutant results in increased interaction between Dpb11 and Mcm2-7 during S phase, impaired GINS interaction with Mcm2-7 during S phase, and decreased replication protein A (RPA) interaction with origin DNA during S phase. We propose a model in which Dpb11 first recruits Cdc45 to Mcm2-7. Dpb11, although bound to Cdc45·Mcm2-7, can block the interaction between GINS and Mcm2-7. Upon extrusion of ssDNA from the central channel of Mcm2-7, Dpb11 dissociates from Mcm2-7, and Dpb11 binds to ssDNA, thereby allowing GINS to bind to Cdc45·Mcm2-7. Finally, we propose that Dpb11 functions with Sld2 and Sld3 to help control the assembly of the replication fork helicase.
Dpb11是芽殖酵母中DNA复制起始所必需的。在S期，Dpb11与S期细胞周期蛋白依赖性激酶磷酸化的Sld2和Sld3结合形成三元复合物。真核生物中的复制叉解旋酶由Cdc45、Mcm2-7和GINS组成。我们在此使用从芽殖酵母中纯化的蛋白质表明，单独的Dpb11与Mcm2-7结合，并且Dpb11还与GINS竞争结合Mcm2-7。此外，Dpb11直接与单链DNA（ssDNA）结合，并且ssDNA抑制Dpb11与Mcm2-7的相互作用。我们还发现Dpb11可以将Cdc45招募到Mcm2-7。我们鉴定出Dpb11的BRCT4基序的一个突变体（dpb11-m1,m2,m3,m5），其在ssDNA存在下仍与Mcm2-7结合，并且当在芽殖酵母细胞中表达时，该突变体表现出DNA复制缺陷。该突变体的表达导致S期Dpb11与Mcm2-7之间的相互作用增加，S期GINS与Mcm2-7之间的相互作用受损，以及S期复制蛋白A（RPA）与起始DNA之间的相互作用减少。我们提出了一个模型，其中Dpb11首先将Cdc45招募到Mcm2-7。Dpb11虽然与Cdc45·Mcm2-7结合，但可以阻断GINS与Mcm2-7之间的相互作用。当ssDNA从Mcm2-7的中央通道挤出时，Dpb11从Mcm2-7解离，并且Dpb11与ssDNA结合，从而允许GINS与Cdc45·Mcm2-7结合。最后，我们提出Dpb11与Sld2和Sld3一起发挥作用，以帮助控制复制叉解旋酶的组装。
