Schulz Laura L, Tyler Jessica K
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Aurora, CO 80045, USA.
FASEB J. 2006 Mar;20(3):488-90. doi: 10.1096/fj.05-5020fje. Epub 2006 Jan 5.
The packaging of the eukaryotic genome into chromatin is likely to regulate all processes that occur on the DNA template. The assembly and disassembly of chromatin structures from histone proteins and DNA are mediated by histone chaperones, including the histone H3/H4 chaperone anti-silencing function 1 (ASF1). To address the function of ASF1 in metazoan cells, we used RNA interference-mediated knockdown of Drosophila melanogaster ASF1 (dASF1). Cells lacking dASF1 accumulate in S phase of the cell cycle as determined by flow cytometry analysis of DNA content and quantitation of the proportion of cells with replication foci. In agreement, bromodeoxyuridine (BrdU) pulse-chase analysis demonstrates that the absence of ASF1 leads to delayed progression through S-phase. Furthermore, the absence of ASF1 leads to a reduced ability to incorporate the nucleoside analog BrdU, indicating that ASF1 is required for efficient DNA replication. We have also found that dASF1 colocalizes with DNA replication foci throughout S phase by immunofluorescence analysis and that these dASF1 foci are disrupted upon inhibition of DNA replication by treatment of cells with hydroxyurea. As such, these results demonstrate that dASF1 is present at active, but not stalled, replication forks. We propose that dASF1 has a direct role in modifying chromatin structure during DNA replication and that this function of dASF1 is important for the processivity of the replication machinery.
真核生物基因组包装成染色质可能会调节发生在DNA模板上的所有过程。染色质结构由组蛋白和DNA组装及拆卸是由组蛋白伴侣介导的，包括组蛋白H3/H4伴侣抗沉默功能1（ASF1）。为了研究ASF1在多细胞动物细胞中的功能，我们使用RNA干扰介导的果蝇ASF1（dASF1）敲低。通过对DNA含量的流式细胞术分析和对具有复制灶的细胞比例的定量分析确定，缺乏dASF1的细胞在细胞周期的S期积累。一致的是，溴脱氧尿苷（BrdU）脉冲追踪分析表明，ASF1的缺失导致S期进程延迟。此外，ASF1的缺失导致掺入核苷类似物BrdU的能力降低，表明ASF1是高效DNA复制所必需的。我们还通过免疫荧光分析发现，dASF1在整个S期与DNA复制灶共定位，并且在用羟基脲处理细胞抑制DNA复制后，这些dASF1灶被破坏。因此，这些结果表明dASF1存在于活跃但未停滞的复制叉处。我们提出，dASF1在DNA复制过程中对染色质结构修饰具有直接作用，并且dASF1的这一功能对复制机器的持续合成能力很重要。
