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Homologous recombination repairs potentially lethal DNA lesions such as double-strand DNA breaks (DSBs) and single-strand DNA gaps (SSGs). In Escherichia coli, DSB repair is initiated by the RecBCD enzyme that resects double-strand DNA ends and loads RecA recombinase to the emerging single-strand (ss) DNA tails. SSG repair is mediated by the RecFOR protein complex that loads RecA onto the ssDNA segment of gaped duplex. In both repair pathways, RecA catalyses reactions of homologous DNA pairing and strand exchange, while RuvABC complex and RecG helicase process recombination intermediates. In this work, we have characterised cytological changes in various recombination mutants of E. coli after three different DNA-damaging treatments: (i) expression of I-SceI endonuclease, (ii) γ-irradiation, and (iii) UV-irradiation. All three treatments caused severe chromosome segregation defects and DNA-less cell formation in the ruvABC, recG, and ruvABC recG mutants. After I-SceI expression and γ-irradiation, this phenotype was efficiently suppressed by the recB mutation, indicating that cytological defects result mostly from incomplete DSB repair. In UV-irradiated cells, the recB mutation abolished cytological defects of recG mutants and also partially suppressed the cytological defects of ruvABC recG mutants. However, neither recB nor recO mutation alone could suppress the cytological defects of UV-irradiated ruvABC mutants. The suppression was achieved only by simultaneous inactivation of the recB and recO genes. Cell survival and microscopic analysis suggest that chromosome segregation defects in UV-irradiated ruvABC mutants largely result from defective processing of stalled replication forks. The results of this study show that chromosome morphology is a valuable marker in genetic analyses of recombinational repair in E. coli. [ABSTRACT FROM AUTHOR]

Abstract: The RecQ helicase is required by the RecF recombination pathway that is operative in recBC(D) sbcB sbcC(D) mutants of Escherichia coli. Genetic data suggest that RecQ participates in resection of DNA ends during initiation of recombination. In vitro, RecQ can unwind a variety of DNA substrates, including recombination intermediates such as D-loops and Holliday junctions. However, its potential role in processing of recombination intermediates during the late stage of the RecF pathway has not been genetically tested. Here we studied the effect of a recQ mutation on transductional recombination and DNA repair after γ-irradiation in ΔrecBCD ΔsbcB sbcC strains deficient for RuvABC, RecG and XerC proteins. RuvABC and RecG proteins process recombination intermediates in the late stage of recombination, whereas XerC is required to resolve chromosome dimers formed upon recombination. Our results do not reveal any substantial synergistic effect between the recQ mutation, on one hand, and ruvABC, recG and xerC mutations on the other. In addition, the recQ mutation suppresses chromosome segregation defects in γ-irradiated ruvABC recG and xerC mutants. These results suggest that RecQ acts upstream of RuvABC, RecG and XerC proteins, a finding that is compatible with its primary role in initiation of the RecF recombination pathway. [Copyright &y& Elsevier]

Human BUBR1 is a 120 kDa protein that plays a central role in the spindle assembly checkpoint (SAC), the evolutionary conserved and self-regulatory system of higher organisms that monitors and repairs defects in chromosome segregation in mitotic cells. BUBR1 is organised into several domains, with an N-terminal region responsible for its localisation into the kinetochore, the multi-component proteinaceous network that assembles onto chromosomes upon mitotic entry. We have expressed and purified uniformly-N/C N-terminal BUBR1 and assigned backbone and side-chain resonances bound to an unlabelled peptide from the protein Blinkin, an element essential for recruitment of BUBR1 to the kinetochore. These assignments provide insights on the Blinkin interaction interface and form the basis of the three-dimensional structure determination of a BUBR1-Blinkin complex. [ABSTRACT FROM AUTHOR]

Background: Despite continuous efforts to identify genes that are pivotal regulators of advanced melanoma and closely related to it, to determine which of these genes have to be blocked in their function to keep this highly aggressive disease in check, it is far from clear which molecular pathway(s) and specific genes therein, is the Achilles' heel of primary and metastatic melanoma. In this report, we present data, which document that the DEAD-box helicase DDX11, which is required for sister chromatid cohesion, is a crucial gatekeeper for melanoma cell survival. Methods: Performing immunohistochemistry and immunoblot analysis, we determined expression of DDX11 in melanoma tissues and cell lines. Following transfection of melanoma cells with a DDX11-specific siRNA, we conducted a qPCR analysis to determine downregulation of DDX11 in the transfected melanoma cells. In subsequent studies, which focused upon an analysis of fluorescently labeled as well as Giesma-stained chromosome spreads, a proliferation analysis and apoptosis assays, we determined the impact of suppressing DDX11 expression on melanoma cells representing advanced melanoma. Result: The findings of the study presented herein document that DDX11 is upregulated with progression from noninvasive to invasive melanoma, and that it is expressed at high levels in advanced melanoma. Furthermore, and equally important, we demonstrate that blocking the expression of DDX11 leads not only to inhibition of melanoma cell proliferation and severe defects in chromosome segregation, but also drives melanoma cells rapidly into massive apoptosis. Conclusion: To date, little is known as to whether helicases play a role in melanoma development and specifically, in the progression from early to advanced melanoma. In this report, we show that the helicase DDX11 is expressed at high levels in primary and metastatic melanoma, and that interfering with its expression leads to severe chromosome segregation defects, telomere shortening, and massive melanoma cell apoptosis. These findings suggest that DDX11 could be an important candidate for molecular targeted therapy for advanced melanoma. [ABSTRACT FROM AUTHOR]

Specific interactions within the cell must occur in a crowded environment and often in a narrow time-space framework to ensure cell survival. In the light that up to 10% of individual protein molecules present at one time in mammalian cells mediate signal transduction, the establishment of productive, specific interactions is a remarkable achievement. The spindle assembly checkpoint (SAC) is an evolutionarily conserved and essential self-monitoring system of the eukaryotic cell cycle that ensures the high fidelity of chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bi-oriented on the mitotic spindle. The function of the SAC involves communication with the kinetochore, an essential multiprotein complex crucial for chromosome segregation that assembles on mitotic or meiotic centromeres to link centromeric DNA with microtubules. Interactions in the SAC and kinetochore-microtubule network often involve the reversible assembly of large multiprotein complexes in which regions of the polypeptide chain that exhibit low structure complexity undergo a disorder-to-order transition. The confinement and high density of protein molecules in the cell has a profound effect on the stability, folding rate, and biological functions of individual proteins and protein assemblies. Here, I discuss the role of large and highly flexible surfaces that mediate productive intermolecular interactions in SAC signaling and postulate that macromolecular crowding contributes to the exquisite regulation that is required for the timely and accurate segregation of chromosomes in higher organisms., (© 2014 Elsevier Inc. All rights reserved.)