| Summary: | 博士 === 國立臺灣大學 === 分子醫學研究所 === 92 === ABSTRACT
The heterodimeric Spt16-Pob3/DUF/FACT complex is a class of chromatin structure modulator with important roles in replication and transcription. Although regarded as transcription elongator for chromatin template, little is known about mammalian FACT’s mode of action and involvement in other molecular processes. Here, we report the identification of a novel interacting and functional partner of FACT, Nek9. Nek9 forms a stable, ~600 kDa complex with FACT in the interphase nuclei. Its active form is characterized by phosphorylation-dependent electrophoretic mobility shift and phosphorylation at a conserved residue within the activation loop (Thr210). Phosphorylation at Thr210 is both necessary and sufficient for the activation of Nek9. When complexed with FACT, Nek9 exhibits markedly elevated phosphorylation on Thr210. Cell cycle analysis on the Nek9dsRNAi cells directly implicated Nek9 in maintaining proper G1 and S progression, a role temporally correlated to the formation of a phospho-Nek9/FACT complex. Collectively, these observations provide evidence that Nek9, potentially as an active enzymatic partner of FACT, mediates certain FACT-associated cellular processes, which are ultimately essential for interphase progression.
Minichromosome maintenance proteins (Mcms), which constitute the proposed replicative helicase, represent another group of cellular factors that associate with the FACT heterodimers. The observation that distinct subassemblies of Mcms form complexes with FACT may suggest their participation in discrete reactions or functions during DNA replication. Furthermore, similar to Nek9, the FACT-associated Mcm4 protein undergoes hyperphosphorylation in a cell cycle-dependent manner, implying a temporal mode of regulation for such complexes. Defect in S-phase progression, potentially at the elongation stage, was observed in yeast cells that harbor conditionally functional versions of Spt16 and Pob3. These results are consistent with the known functions of Mcm proteins in DNA replication and further strengthen the link of FACT to this chromatin-based molecular process.
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