We have comprehensively mapped long-range associations between chromosomal regions throughout the fission yeast genome using the latest genomics approach that combines next generation sequencing and chromosome conformation capture (3C). Our relatively simple approach, referred to as enrichment of ligation products (ELP), involves digestion of the 3C sample with a 4 bp cutter and self-ligation, achieving a resolution of 20 kb. It recaptures previously characterized genome organizations and also identifies new and important interactions. We have modeled the 3D structure of the entire fission yeast genome and have explored the functional relationships between the global genome organization and transcriptional regulation. We find significant associations among highly transcribed genes. Moreover, we demonstrate that genes co-regulated during the cell cycle tend to associate with one another when activated. Remarkably, functionally defined genes derived from particular gene ontology groups tend to associate in a statistically significant manner. Those significantly associating genes frequently contain the same DNA motifs at their promoter regions, suggesting that potential transcription factors binding to these motifs are involved in defining the associations among those genes. Our study suggests the presence of a global genome organization in fission yeast that is functionally similar to the recently proposed mammalian transcription factory.

The authors show that Pol III transcribed genes such as tRNA and 5S rRNA genes localize to centromeres in fission yeast. The centromeric localization of Pol III genes is mediated by condensin. This study suggests that there is a functional link between the centromeric localization of dispersed Pol III genes and mitotic chromosome condensation.

YddV from Escherichia coli (Ec) is a novel globin-coupled heme-based oxygen sensor protein displaying diguanylate cyclase activity in response to oxygen availability. In this study, we quantified the turnover numbers of the active [Fe(III), 0.066 min(-1); Fe(II)-O(2) and Fe(II)-CO, 0.022 min(-1)] [Fe(III), Fe(III)-protoporphyrin IX complex; Fe(II), Fe(II)-protoporphyrin IX complex] and inactive forms [Fe(II) and Fe(II)-NO, <0.01 min(-1)] of YddV for the first time. Our data indicate that the YddV reaction is the rate-determining step for two consecutive reactions coupled with phosphodiesterase Ec DOS activity on cyclic di-GMP (c-di-GMP) [turnover number of Ec DOS-Fe(II)-O(2), 61 min(-1)]. Thus, O(2) binding and the heme redox switch of YddV appear to be critical factors in the regulation of c-di-GMP homeostasis. The redox potential and autoxidation rate of heme of the isolated heme domain of YddV (YddV-heme) were determined to be -17 mV versus the standard hydrogen electrode and 0.0076 min(-1), respectively. The Fe(II) complexes of Y43A and Y43L mutant proteins (residues at the heme distal side of the isolated heme-bound globin domain of YddV) exhibited very low O(2) affinities, and thus, their Fe(II)-O(2) complexes were not detected on the spectra. The O(2) dissociation rate constant of the Y43W protein was >150 s(-1), which is significantly larger than that of the wild-type protein (22 s(-1)). The autoxidation rate constants of the Y43F and Y43W mutant proteins were 0.069 and 0.12 min(-1), respectively, which are also markedly higher than that of the wild-type protein. The resonance Raman frequencies representing ν(Fe-O(2)) (559 cm(-1)) of the Fe(II)-O(2) complex and ν(Fe-CO) (505 cm(-1)) of the Fe(II)-CO complex of Y43F differed from those (ν(Fe-O(2)), 565 cm(-1); ν(Fe-CO), 495 cm(-1)) of the wild-type protein, suggesting that Tyr43 forms hydrogen bonds with both O(2) and CO molecules. On the basis of the results, we suggest that Tyr43 located at the heme distal side is important for the O(2) recognition and stability of the Fe(II)-O(2) complex, because the hydroxyl group of the residue appears to interact electrostatically with the O(2) molecule bound to the Fe(II) complex in YddV. Our findings clearly support a role of Tyr in oxygen sensing, and thus modulation of overall conversion from GTP to pGpG via c-di-GMP catalyzed by YddV and Ec DOS, which may be applicable to other globin-coupled oxygen sensor enzymes.

Heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) is a gas-sensor enzyme that hydrolyzes cyclic dinucleotide-GMP, and it is activated by O 2 or CO binding to the Fe(II) heme. In contrast to other well known heme-regulated gas-sensor enzymes or proteins, Ec DOS is not specific for a single gas ligand. Because Arg 97 in the heme distal side in Ec DOS interacts with the O 2 molecule and Met 95 serves as the axial ligand on the distal side of the Fe(II) heme-bound PAS domain of Ec DOS, we explored the effect of mutating these residues on the activity and gas specificity of Ec DOS. We found that R97A, R97I, and R97E mutations do not significantly affect regulation of the phosphodiesterase activities of the Fe(II)-CO and Fe(II)-NO complexes. The phosphodiesterase activities of the Fe(II)-O 2 complexes of the mutants could not be detected due to rapid autoxidation and/or low affinity for O 2 . In contrast, the activities even of the gas-free M95A and M95L mutants were similar to that of the gas-activated wild-type enzyme. Interestingly, the activity of the M95H mutant was partially activated by O 2 , CO, and NO. Spectroscopic analysis indicated that the Fe(II) heme is in the 5-coordinated high-spin state in the M95A and M95L mutants but that in the M95H mutant, like wild-type Ec DOS, it is in the 6-coordinated low-spin state. These results suggest that Met 95 coordination to the Fe(II) heme is critical for locking the system and that global structural changes around Met 95 caused by the binding of the external ligands or mutations at Met 95 releases the catalytic lock and activates catalysis.Cyclic dinucleotide-GMP (c-diGMP) 2 is a novel intracellular second messenger that regulates cell motility, differentiation, development, virulence, antibiotic formation, and biofilm formation in bacteria growth and factor-stimulated proliferation in human colon cancer cells (1-10). Enzymes involved in the biosynthesis and breakdown of c-diGMP contain highly homologous GGDEF or EAL subdomains, respectively (1-10). The GGDEF subdomain expresses diguanylate cyclase activity, and catalyzes the synthesis of one molecule of cyclic diGMP from two molecules of GTP via the linear intermediate diguanosine tetraphosphate. The GGDEF subdomain is ϳ180 amino acids long and has a conserved amino acid sequence, GG(D/E)(D/E)F. The EAL subdomain has a phosphodiesterase activity that hydrolytically cleaves cyclic diGMP into l-diGMP and/or GMP, and is 260 residues in length, including the conserved amino acid sequence EAL. Metabolism of c-diGMP may be physiologically important because the genome of Escherichia coli K-12, for example, encodes 19 proteins with GGDEF subdomains and 17 with EAL subdomains.Heme-regulated phosphodiesterase from E. coli (Ec DOS) contains a heme-bound PAS domain in the N-terminal region and a phosphodiesterase domain with GGDEF and EAL subdomains in the C-terminal region (11, 12). Although Ec DOS also contains a GGDEF subdomain, it does not appear to have guanylate cyclase activity. Thus, the precise roles of t...

SUMMARY Complex genome organizations participate in various nuclear processes including transcription, DNA replication and repair. However, the mechanisms that generate and regulate these functional genome structures remain largely unknown. Here, we describe how the Ku heterodimer complex, which functions in nonhomologous end joining, mediates clustering of long terminal repeat (LTR) retrotransposons at centromeres in fission yeast. We demonstrate that the CENP-B subunit, Abp1, functions as a recruiter of the Ku complex, which in turn loads the genome-organizing machinery condensin to retrotransposons. Intriguingly, histone H3 Lys56 (H3K56) acetylation, which functions in DNA replication and repair, interferes with Ku localization at retrotransposons without disrupting Abp1 localization and, as a consequence, dissociates condensin from retrotransposons. This dissociation releases condensin-mediated genomic associations during S phase and upon DNA damage. ATR (ATM and Rad3-related) kinase mediates DNA damage-response of condensin-mediated genome organization. Our study describes a function of H3K56 acetylation that neutralizes condensin-mediated genome organization.