The initiation of Escherichia coli chromosomal DNA replication starts with the oligomerization of the DnaA protein at repeat sequences within the origin (ori) region. The amount of ori DNA per cell directly correlates with the growth rate. During fast growth, the cell generation time is shorter than the time required for complete DNA replication; therefore, overlapping rounds of chromosome replication are required. Under these circumstances, the ori region DNA abundance exceeds the DNA abundance in the termination (ter) region. Here, high ori/ter ratios are found to persist in (p)ppGpp-deficient [(p)ppGpp0] cells over a wide range of balanced exponential growth rates determined by medium composition. Evidently, (p)ppGpp is necessary to maintain the usual correlation of slow DNA replication initiation with a low growth rate. Conversely, ori/ter ratios are lowered when cell growth is slowed by incrementally increasing even low constitutive basal levels of (p)ppGpp without stress, as if (p)ppGpp alone is sufficient for this response. There are several previous reports of (p)ppGpp inhibition of chromosomal DNA synthesis initiation that occurs with very high levels of (p)ppGpp that stop growth, as during the stringent starvation response or during serine hydroxamate treatment. This work suggests that low physiological levels of (p)ppGpp have significant functions in growing cells without stress through a mechanism involving negative supercoiling, which is likely mediated by (p)ppGpp regulation of DNA gyrase. IMPORTANCE Bacterial cells regulate their own chromosomal DNA synthesis and cell division depending on the growth conditions, producing more DNA when growing in nutritionally rich media than in poor media (i.e., human gut versus water reservoir). The accumulation of the nucleotide analog (p)ppGpp is usually viewed as serving to warn cells of impending peril due to otherwise lethal sources of stress, which stops growth and inhibits DNA, RNA, and protein synthesis. This work importantly finds that small physiological changes in (p)ppGpp basal levels associated with slow balanced exponential growth incrementally inhibit the intricate process of initiation of chromosomal DNA synthesis. Without (p)ppGpp, initiations mimic the high rates present during fast growth. Here, we report that the effect of (p)ppGpp may be due to the regulation of the expression of gyrase, an important enzyme for the replication of DNA that is a current target of several antibiotics.
Background/Aim: Androgens are essential for the growth of most prostate cancers (PCa). As a result, androgen ablation is the mainstay of the treatment of PCa. Proteins of the polycomb and trithorax family are master epigenetic regulators of cell type specific gene expression including androgen receptor. Materials and Methods: We interrogated epigenetic changes of a 24-gene panel corresponding to polycomb and trithorax genes by PCR array and differential gene expression by quantitative real time-PCR on prostate cancer cell line (LNCaP) treated with the synthetic ligand R1881. Results: We observed the highest methylation for CBX2, PCGF6, PHC2, EZH2 and TRIM27 genes and the lowest methylation for CBX8 and PCGF2 (p<0.05), and a modest decrease in the expression of EZH2. Conclusion: Differential methylation profiles of polycomb and trithorax genes may contribute to the dynamics of prostate carcinogenesis.
The guanosine nucleotide derivatives ppGpp and pppGpp are central to the remarkable capacity of bacteria to adapt to fluctuating environment and metabolic perturbations. These alarmones are synthesized by two proteins, RelA and SpoT in E. coli and the activities of each of the two enzymes are highly regulated for homeostatic control of (p)ppGpp levels in the cell. Although the domain structure and function of RelA are well defined, the findings of this study unfold the regulatory aspect of RelA that is possibly relevant in vivo. We uncover here the importance of the N-terminal 1-119 amino acids of the enzymatically compromised (p)ppGpp hydrolytic domain (HD) of monofunctional RelA for the (p)ppGpp mediated regulation of RelA-CTD function. We find that even moderate level expression of RelA appreciably reduces growth when the basal levels of (p)ppGpp in the cells are higher than in the wild type, an effect independent of its ability to synthesize (p)ppGpp. This is evidenced by the growth inhibitory effects of oversynthesis of the RelA-CTD in the relA+ strain but not in relA null mutant, suggesting the requirement of the functional RelA protein for basal level synthesis of (p)ppGpp, accordingly corroborated by the restoration of the growth inhibitory effects of the RelA-CTD expression in the relA1 spoT202 mutant. The N-terminal 119 amino acids of RelA fused in-frame with the RelA-CTD, both from 406-744 amino acids (including TGS) and from 454-744 amino acids (sans TGS) caused growth inhibition only in spoT1 and spoT202 relA1 mutants, uncovering the hitherto unrealized (p)ppGpp-dependent regulation of RelA-CTD function. An incremental rise in the (p)ppGpp levels is proposed to progressively modulate the interaction of RelA-CTD with the ribosomes, with possible implications in the feedback regulation of the N-terminal (p)ppGpp synthesis function, a proposal that best explains the nonlinear relationship between (p)ppGpp synthesis and increased ratio of RelA:ribosomes, both in vitro as well as in vivo.
The RAS protein activator like 2 (RASAL2) negatively regulates RAS proto-oncogene which is activated by high mutation rate in cancer. Thus, RASAL2 expression could potentially limit the function of RAS in prostate cancer (PCa). Genome-wide DNA methylation analysis demonstrated that RASAL2 is differentially hypermethylated in PCa tissues compared to benign prostate tissues. The PCR analysis of RASAL2 mRNA transcript showed differential expression in a panel of prostate cell lines with most PCa showing lower RASAL2 expression compared to benign prostatic epithelial cells. In PCa PC3 cells, the ectopic expression of RASAL2 significantly inhibited cell proliferation and invasion and induced an S phase plus G2/M phase cell cycle arrest. Ingenuity Pathway Analysis (IPA) demonstrated a cross talk between RASAL2 and TNFα, a key cytokine in immune signaling pathway that is relevant in PCa. Over-expression of RASAL2 downregulated TNFα expression whereas the knockdown of RASAL2 caused increased expression of TNFα. Taken together, our data demonstrates tumor suppressor role for RASAL2 in human PCa cells, despite increased RAS oncogenic activity. Our observation provides a new mechanistic insight of RASAL2 expression in aberrant Ras expression and immune signaling in PCa cells suggesting a potential novel therapeutic target for PCa.
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