Bruck Irina, Dhingra Nalini, Martinez Matthew P, Kaplan Daniel L
Florida State University College of Medicine, Department of Biomedical Sciences, Tallahassee, Florida, United States of America.
PLoS One. 2017 May 3;12(5):e0177147. doi: 10.1371/journal.pone.0177147. eCollection 2017.
Dpb11 is required for the initiation of DNA replication in budding yeast. We found that Dpb11 binds tightly to single-stranded DNA (ssDNA) or branched DNA structures, while its human homolog, TopBP1, binds tightly to branched-DNA structures. We also found that Dpb11 binds stably to CDK-phosphorylated RPA, the eukaryotic ssDNA binding protein, in the presence of branched DNA. A Dpb11 mutant specifically defective for DNA binding did not exhibit tight binding to RPA in the presence of DNA, suggesting that Dpb11-interaction with DNA may promote the recruitment of RPA to melted DNA. We then characterized a mutant of Dpb11 that is specifically defective in DNA binding in budding yeast cells. Expression of dpb11-m1,2,3,5,ΔC results in a substantial decrease in RPA recruitment to origins, suggesting that Dpb11 interaction with DNA may be required for RPA recruitment to origins. Expression of dpb11-m1,2,3,5,ΔC also results in diminished GINS interaction with Mcm2-7 during S phase, while Cdc45 interaction with Mcm2-7 is like wild-type. The reduced GINS interaction with Mcm2-7 may be an indirect consequence of diminished origin melting. We propose that the tight interaction between Dpb11, CDK-phosphorylated RPA, and branched-DNA may be required for the essential function of stabilizing melted origin DNA in vivo. We also propose an alternative model, wherein Dpb11-DNA interaction is required for some other function in DNA replication initiation, such as helicase activation.
Dpb11是芽殖酵母中DNA复制起始所必需的。我们发现Dpb11与单链DNA（ssDNA）或分支DNA结构紧密结合，而其人类同源物TopBP1则与分支DNA结构紧密结合。我们还发现，在分支DNA存在的情况下，Dpb11与CDK磷酸化的真核ssDNA结合蛋白RPA稳定结合。一个在DNA结合方面存在特异性缺陷的Dpb11突变体在DNA存在时未表现出与RPA的紧密结合，这表明Dpb11与DNA的相互作用可能促进RPA募集到解链的DNA上。然后我们对芽殖酵母细胞中在DNA结合方面存在特异性缺陷的Dpb11突变体进行了表征。dpb11-m1,2,3,5,ΔC的表达导致RPA募集到起始位点的数量大幅减少，这表明Dpb11与DNA的相互作用可能是RPA募集到起始位点所必需的。dpb11-m1,2,3,5,ΔC的表达还导致S期GINS与Mcm2-7的相互作用减弱，而Cdc45与Mcm2-7的相互作用则与野生型相似。GINS与Mcm2-7相互作用的减弱可能是起始位点解链减少的间接结果。我们提出，Dpb11、CDK磷酸化的RPA和分支DNA之间的紧密相互作用可能是在体内稳定解链的起始位点DNA这一基本功能所必需的。我们还提出了另一种模型，即Dpb11与DNA的相互作用是DNA复制起始中某些其他功能（如解旋酶激活）所必需的。
