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Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis , the nucleoid occlusion protein Noc protects the chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP‐on‐Chip and bioinformatics, we have identified a consensus Noc‐binding DNA sequence (NBS), and have shown that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus. Purified Noc bound specifically to an NBS in vitro . NBSs inserted near the replication terminus bound Noc–YFP and caused a delay in cell division. An autonomous plasmid carrying an NBS array recruited Noc–YFP and conferred a severe Noc‐dependent inhibition of cell division. This shows that Noc is a potent inhibitor of division, but that its activity is strictly localized by the interaction with NBS sites in vivo . We propose that Noc serves not only as a spatial regulator of cell division to protect the nucleoid, but also as a timing device with an important role in the coordination of chromosome segregation and cell division.

Nucleus accumbens-associated protein 1 (NAC1) is a nuclear protein that harbors an amino-terminal BTB domain and a carboxyl-terminal BEN domain. NAC1 appears to play significant and diverse functions in cancer and stem cell biology. Here we demonstrated that the BEN domain of NAC1 is a sequence-specific DNA-binding domain. We selected the palindromic 6 bp motif ACATGT as a target sequence by using a PCR-assisted random oligonucleotide selection approach. The interaction between NAC1 and target DNA was characterized by gel shift assays, pull-down assays, isothermal titration calorimetry (ITC), chromatin-immunoprecipitation assays, and NMR chemical shifts perturbation (CSP). The solution NMR structure revealed that the BEN domain of human NAC-1 is composed of five conserved α helices and two short β sheets, with an additional hitherto unknown N-terminal α helix. In particular, ITC clarified that there are two sequential events in the titration of the BEN domain of NAC1 into the target DNA. The ITC results were further supported by CSP data and structure analyses. Furthermore, live cell photobleaching analyses revealed that the BEN domain of NAC1 alone was unable to interact with chromatin/other proteins in cells.

UL34 is one of the ~50 genes of human cytomegalovirus (HCMV) required for replication in cell culture in human fibroblasts. UL34 encodes highly related early (UL34a) and late (UL34b) proteins that are virtually identical, with the early protein containing an additional 21 amino terminal amino acids. The UL34 proteins are sequence-specific DNA‑binding proteins that localize to the nucleus. The HCMV genome contains 14 to 15 UL34 binding sites; two of the UL34 binding sites contribute to transcriptional regulation of two other viral genes, US3 and US9. The roles of the remaining binding sites and the requirement for both UL34 proteins during viral infection remain unknown. We examined the contributions of the early and late UL34 proteins to viral replication by generating HCMV-containing bacterial artificial chromosomes with the initiation codon for the early or the late protein mutated. Neither virus was able to replicate, demonstrating that UL34 expression is required throughout the viral replication cycle. A marked decrease in viral gene expression for each of the mutants suggests that UL34 proteins may contribute generally to transcriptional regulation. Intracellular localization studies demonstrated that UL34 colocalizes with the major immediate early protein, IE2, and the viral DNA polymerase processivity factor, UL44, to viral DNA replication centers. In conclusion, sustained UL34 protein expression is required for viral replication. The sequence-specific DNA binding ability of UL34 proteins, their localization to viral DNA replication centers and their general effects on viral gene expressions suggests that UL34 proteins contribute to the establishment of a nuclear environment necessary for viral gene expression and DNA replication.

Organization of replicating prokaryotic genomes requires architectural elements that, similarly to eukaryotic systems, induce topological changes such as DNA supercoiling. Bacteriophage Φ29 protein p6 has been described as a histone-like protein that compacts the viral genome by forming a nucleoprotein complex and plays a key role in the initiation of protein-primed DNA replication. In this work, we analyze the subcellular localization of protein p6 by immunofluorescence microscopy and show that, at early infection stages, it localizes in a peripheral helix-like configuration. Later, at middle infection stages, protein p6 is recruited to the bacterial nucleoid. This migrating process is shown to depend on the synthesis of components of the Φ29 DNA replication machinery (i.e., terminal protein and DNA polymerase) needed for the replication of viral DNA, which is required to recruit the bulk of protein p6. Importantly, the double-stranded DNA-binding capacity of protein p6 is essential for its relocalization at the nucleoid. Altogether, the results disclose the in vivo organization of a viral histone-like protein in bacteria.

In this review we describe the multiple functions of p53 in response to DNA damage, with an emphasis on p53's role in DNA repair. We summarize data demonstrating that p53, through its various biochemical activities and via its ability to interact with components of the repair and recombination machinery, actively participates in various processes of DNA repair and DNA recombination. An important aspect in evaluating p53 functions arises from the finding that the p53 core domain harbors two mutually exclusive biochemical activities, sequence-specific DNA binding, required for its transactivation function, and 3'->5' exonuclease activity, possibly involved in various aspects of DNA repair. As modifications of p53 that lead to activation of its sequence-specific DNA-binding activity result in inactivation of its 3'-> 5' exonuclease activity, we propose that p53 exerts its functions as a 'guardian of the genome' at various levels: in its non-induced state, p53 should not be regarded as a non-functional protein, but might be actively involved in prevention and repair of endogenous DNA damage, for example via its exonuclease activity. Upon induction through exogenous DNA damage, p53 will exert its well-documented functions as a superior response element in various types of cellular stress. The dual role model for p53 in maintaining genomic integrity significantly enhances p53's possibilities as a guardian of the genome.

The tumour suppressor p53 is a potent mediator of cellular responses against genotoxic insults. In this review we describe the multiple functions of p53 in response to DNA damage, with an emphasis on p53's role in DNA repair. We summarize data demonstrating that p53 actively participates in various processes of DNA repair and DNA recombination via its ability to interact with components of the repair and recombination machinery, and by its various biochemical activities. An important aspect in evaluating p53 functions is provided by the finding that the core domain of p53 harbours two mutually exclusive biochemical activities, sequence-specific DNA binding required for its transactivation function, and 3′–5′ exonuclease activity, possibly involved in aspects of DNA repair. Based on the finding that modifications of p53 which lead to activation of its sequence-specific DNA-binding activity result in inactivation of its 3′–5′ exonuclease activity, we propose that p53 exerts its functions as a ‘guardian of the genome’ at various levels in its noninduced state, p53 should not be regarded as a ‘dead’ protein but, for example, via its exonuclease activity might be actively involved in prevention and repair of endogenous DNA damage. Upon induction through exogenous DNA damage, p53 will exert its well-documented functions as a superior response element in various types of cellular stress. This dual role model for p53 in maintaining genomic integrity significantly enhances p53's possibilities as a guardian of the genome.

The most import biological function of the tumor suppressor p53 is that of a sequence-specific transactivator. In response to a variety of cellular stress stimuli, p53 induces the transcription of an ever-increasing number of target genes, leading to growth arrest and repair, or to apoptosis. Long considered as a \"latent\" DNA binder that requires prior activation by C-terminal modification, recent data provide strong evidence that the DNA binding activity of p53 is strongly dependent on structural features within the target DNA and is latent only if the target DNA lacks a certain structural signal code. In this review we discuss evidence for complex interactions of p53 with DNA, which are strongly dependent on the dynamics of DNA structure, especially in the context of chromatin. We provide a model of how this complexity may serve to achieve selectivity of target gene regulation by p53 and how DNA structure in the context of chromatin may serve to modulate p53 functions.Key words: tumor suppressor p53, sequence-specific DNA binding, DNA conformation, chromatin, chromatin remodeling.

Transcriptional activation by the tumor suppressor p53 is regulated at multiple levels, including posttranslational modifications of the p53 protein, interaction of p53 with various regulatory proteins, or at the level of sequence-specific DNA binding to the response elements in p53's target genes. We here propose as an additional regulatory mechanism that the DNA topology of p53-responsive promoters may determine the interaction of p53 with its target genes. We demonstrate that sequence-specific DNA binding (SSDB) and transcriptional activation by p53 of the mdm2 promoter is inhibited when this promoter is present in supercoiled DNA, where it forms a non-B-DNA structure which spans the p53-responsive elements. Relaxation of the supercoiled DNA in vitro resulted in conversion of the non-B-DNA to a B-DNA conformation within the mdm2 promoter, and correlated with an enhanced SSDB of p53 and an elevated expression of a reporter gene. In contrast, sequence specific DNA binding and transcriptional activation of the p21 promoter were not inhibited by DNA supercoiling. We propose that conformational alterations within p53-responsive sites, which either promote or prohibit sequence specific DNA binding of p53, are an important feature in orchestrating the activation of different p53 responsive promoters.

The post-genomic era has ushered in the extensive application of epigenetic editing tools, allowing for precise alterations of gene expression. The use of reprogrammable editors that carry transcriptional corepressors has significant potential for long-term epigenetic silencing for the treatment of human diseases. The ideal scenario involves precise targeting of a specific genomic location by a DNA-binding domain, ensuring there are no off-target effects and that the process yields no genetic remnants aside from specific epigenetic modifications (i.e., DNA methylation). A notable example is a recent study on the mouse Pcsk9 gene, crucial for cholesterol regulation and expressed in hepatocytes, which identified synthetic zinc-finger (ZF) proteins as the most effective DNA-binding editors for silencing Pcsk9 efficiently, specifically, and persistently. This discussion focuses on enhancing the specificity of ZF-array DNA binding by optimizing interactions between specific amino acids and DNA bases across three promoters containing CpG islands.

The binding specificities of RNA- and DNA-binding proteins are determined from experimental data using a ‘deep learning’ approach. Knowing the sequence specificities of DNA- and RNA-binding proteins is essential for developing models of the regulatory processes in biological systems and for identifying causal disease variants. Here we show that sequence specificities can be ascertained from experimental data with 'deep learning' techniques, which offer a scalable, flexible and unified computational approach for pattern discovery. Using a diverse array of experimental data and evaluation metrics, we find that deep learning outperforms other state-of-the-art methods, even when training on in vitro data and testing on in vivo data. We call this approach DeepBind and have built a stand-alone software tool that is fully automatic and handles millions of sequences per experiment. Specificities determined by DeepBind are readily visualized as a weighted ensemble of position weight matrices or as a 'mutation map' that indicates how variations affect binding within a specific sequence.