RESUMO
DNA origami provides a versatile platform for conducting 'architecture-function' analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis.
Assuntos
DNA Fúngico/isolamento & purificação , DNA de Cadeia Simples/química , Hibridização de Ácido Nucleico/métodos , Oligonucleotídeos/química , DNA Fúngico/química , DNA Fúngico/genética , DNA de Cadeia Simples/genética , Glutationa/química , Glicerol/química , Cinetocoros/química , Lasers , Microscopia Eletrônica , Proteínas Nucleares/química , Proteínas Nucleares/genética , Oligonucleotídeos/genética , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Leveduras/genéticaRESUMO
During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression.
Assuntos
Cinesinas/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Motores Moleculares , Mutação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Accurate segregation of duplicated chromosomes ensures that daughter cells get one and only one copy of each chromosome. Errors in chromosome segregation result in aneuploidy and have severe consequences on human health. Incorrect chromosome number and chromosomal instability are hallmarks of tumor cells. Hence, segregation errors are thought to be a major cause of tumorigenesis. A study of the physical mechanical basis of chromosome segregation is essential to understand the processes that can lead to errors. Tremendous progress has been made in recent years in identifying the proteins necessary for chromosome movement and segregation, but the mechanism and structure of critical force generating components and the molecular basis of centromere stiffness remain poorly understood.