RESUMO
Structural maintenance of chromosomes (SMC) protein complexes, including cohesin and condensin, play key roles in the regulation of higher-order chromosome organization. Even though SMC proteins are thought to mechanistically determine the function of the complexes, their native conformations and dynamics have remained unclear. Here, we probe the topology of Smc2-Smc4 dimers of the S. cerevisiae condensin complex with high-speed atomic force microscopy (AFM) in liquid. We show that the Smc2-Smc4 coiled coils are highly flexible polymers with a persistence length of only â¼ 4 nm. Moreover, we demonstrate that the SMC dimers can adopt various architectures that interconvert dynamically over time, and we find that the SMC head domains engage not only with each other, but also with the hinge domain situated at the other end of the â¼ 45-nm-long coiled coil. Our findings reveal structural properties that provide insights into the molecular mechanics of condensin complexes.
Assuntos
Proteínas de Transporte/química , Proteínas Cromossômicas não Histona/química , Cromossomos Fúngicos/química , Proteínas Nucleares/química , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/ultraestrutura , Expressão Gênica , Processamento de Imagem Assistida por Computador , Microscopia de Força Atômica/métodos , Simulação de Dinâmica Molecular , Imagem Molecular , Método de Monte Carlo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Domínios Proteicos , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
We use Monte Carlo simulations to analyze the simultaneous interactions of multiple proteins to a long DNA molecule. We study the time dependence of protein organization on DNA for different regimes that comprise (non)cooperative sequence-independent protein assembly, dissociation, and linear motion. A range of different behaviors is observed for the dynamics, final coverage, and cluster size distributions. We observe that the DNA substrate is almost never completely covered by protein when taking into account only (non)cooperative binding, because gaps remain on the substrate that are smaller than the binding site size of the protein. Due to these gaps, the apparent binding size of a protein during noncooperative binding can be overestimated by up to 30%. During dissociation of cooperatively bound proteins, the dissociation curve can be exponentially shaped even when allowing only end-dependent dissociation. We discuss the potential of our method for the analysis of a number of single-molecule experiments, for example, the binding of the DNA-repair proteins RecA and Rad51 to DNA.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/ultraestrutura , DNA/química , DNA/ultraestrutura , Modelos Químicos , Modelos Moleculares , Sítios de Ligação , Simulação por Computador , Modelos Estatísticos , Método de Monte Carlo , Movimento (Física) , Ligação ProteicaRESUMO
The human DNA repair protein RAD51 is the crucial component of helical nucleoprotein filaments that drive homologous recombination. The molecular mechanistic details of how this structure facilitates the requisite DNA strand rearrangements are not known but must involve dynamic interactions between RAD51 and DNA. Here, we report the real-time kinetics of human RAD51 filament assembly and disassembly on individual molecules of both single- and double-stranded DNA, as measured using magnetic tweezers. The relative rates of nucleation and filament extension are such that the observed filament formation consists of multiple nucleation events that are in competition with each other. For varying concentration of RAD51, a Hill coefficient of 4.3 +/- 0.5 is obtained for both nucleation and filament extension, indicating binding to dsDNA with a binding unit consisting of multiple (> or =4) RAD51 monomers. We report Monte Carlo simulations that fit the (dis)assembly data very well. The results show that, surprisingly, human RAD51 does not form long continuous filaments on DNA. Instead each nucleoprotein filament consists of a string of many small filament patches that are only a few tens of monomers long. The high flexibility and dynamic nature of this arrangement is likely to facilitate strand exchange.