Dynamic changes in chromatin structure are essential for efficient DNA processing such as transcription, replication, and DNA repair. Histone modifications and ATP-dependent chromatin remodeling are important for the alteration of chromatin structure. The INO80 chromatin remodeling complex plays an important role in HR-mediated repair of DNA double-strand breaks (DSBs). In yeast, the INO80 complex is recruited to the sites of DSBs via direct interaction with phosphorylated histone H2A and facilitates the processing of DSB ends. However, the function of the mammalian INO80 complex in DNA repair is mostly unknown. Here, we show that the mammalian INO80 complex is recruited to the laser-induced DNA damage sites in a phosphorylated H2AX (γH2AX)-independent manner. We also found that an actin-related protein, ARP8, is an important subunit that is required for the recruitment of the mammalian INO80 complex to the DNA damage sites, although the recruitment of the yeast INO80 complex requires its Nhp10 or Arp4 subunits. These results suggest that the mammalian INO80 complex is also recruited to DNA damage sites similarly to the yeast INO80 complex, but the mechanism of this recruitment may be different from that of the yeast INO80 complex. These findings provide new insights into the mechanisms of DNA repair in mammalian cells.
The yeast Dbp9p is a member of the DEAD box family of RNA helicases, which are thought to be involved in RNA metabolism. Dbp9p seems to function in ribosomal RNA biogenesis, but it has not been biochemically characterized. To analyze the enzymatic characteristics of the protein, we expressed a recombinant Dbp9p in Escherichia coli and purified it to homogeneity. The purified protein exhibited RNA unwinding and binding activity in the absence of NTP, and this activity was abolished by a mutation in the RNA-binding domain. We then characterized the ATPase activity of Dbp9p with respect to cofactor specificity; the activity was found to be severely inhibited by yeast total RNA and moderately inhibited by poly(U), poly(A), and poly(C) but to be stimulated by yeast genomic DNA and salmon sperm DNA. In addition, Dbp9p exhibited DNA-DNA and DNA-RNA helicase activity in the presence of ATP. These results indicate that Dbp9p has biochemical characteristics unique among DEAD box proteins.RNA helicases are enzymes that unwind double-stranded RNA molecules in an energy-dependent manner through the hydrolysis of NTP. These proteins are widely distributed among a variety of organisms ranging from viruses and prokaryotes to mammals. RNA helicases are associated with virtually all biological processes requiring RNA, including transcription, splicing, RNA transport, ribosome biogenesis, RNA editing, translation, and RNA decay (1). The largest family of RNA helicases is the DEAD box protein family.The DEAD box proteins have seven to eight distinctive motifs. The DEAD is derived from the amino acid sequence of motif II, the Walker B motif (2, 3). In vitro analyses of DEAD box proteins such as the translation initiation factor eIF-4A and the human nuclear protein p68 have demonstrated that these proteins possess RNA-dependent ATPase activity and are capable of melting short RNA duplex structures in an ATP-dependent manner (3-5). For example, eIF-4A, an archetypical member of the DEAD box protein family, is capable of unwinding partial duplex RNA in a bidirectional manner and acting on RNA or DNA-RNA, but not on the DNA duplex (4, 6). Extensive mutational analyses of the conserved regions of DEAD box proteins have demonstrated that these regions are important to ATP binding, ATP hydrolysis, RNA binding, RNA unwinding, and coupling of these different activities. In addition to these typical DEAD box RNA helicases, some DEAD box proteins have recently been shown to have peculiar characteristics. Hepatitis C virus NS3 drives the unwinding activity with all ribo-and deoxyribo-NTPs (6). Moreover, CsdA, an Escherichia coli DEAD box protein, unwinds double-stranded RNA in the absence of NTP (7). These reports suggest that the DEAD box family includes proteins with a wide variety of biochemical activities.The yeast Saccharomyces cerevisiae contains over 20 different DEAD box proteins, many of which are essential to viability. Combined with biochemical analyses, yeast genetical analyses have revealed the functions of many DEAD box proteins. ...
Transcriptional activation and repression are a key step in the regulation of all cellular activities. The development of comprehensive analysis methods such as DNA microarray has advanced our understanding of the correlation between the regulation of transcription and that of cellular mechanisms. However, DNA microarray analysis based on steady-state mRNA (total mRNA) does not always correspond to transcriptional activation or repression. To comprehend these transcriptional regulations, the detection of nascent RNAs is more informative. Although the nuclear run-on assay can detect nascent RNAs, it has not been fully applied to DNA microarray analysis. In this study, we have developed a highly efficient method for isolating bromouridine-labeled nascent RNAs that can be successfully applied to DNA microarray analysis. This method can linearly amplify small amounts of mRNAs with little bias. Furthermore, we have applied this method to DNA microarray analysis from mouse G2-arrested cells and have identified several genes that exhibit novel expression profiles. This method will provide important information in the field of transcriptome analysis of various cellular processes.
During mammalian mitosis, transcription is silenced due to dissociation of transcription factors from DNA and chromosome condensation. At the end of mitosis, transcription is reactivated through chromosome relaxation and reloading of these factors to the DNA. Early G1 genes, which are preferentially reactivated during M/G1 transition, are important for maintenance of cellular function and are known to be strictly regulated. As only few early G1 genes have been identified to date, screening for early G1 genes by genome-wide analysis using nascent mRNA could contribute to the elucidation of the regulatory mechanisms during early G1. Here, we performed a detailed expression analysis for the M/G1 transition of mammalian cells by microarray analysis of nascent mRNA, and identified 298 early G1 genes. Analysis of these genes provides two important insights. Firstly, certain motifs are enriched in the upstream sequences of early G1 genes; from this we could predict candidate cognate transcription factors, including Sp1, which is recruited to the DNA in the early G1 phase. Secondly, we discovered that neighboring genes of early G1 genes were also frequently up-regulated in the G1 phase. Information about these numerous newly identified early G1 genes will likely contribute to an understanding of the regulatory mechanisms of the early G1 genes.
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