Protocol for studying topological DNA interactions by purified fission yeast condensin.

To understand the transition from interphase chromatin into well-shaped chromosomes during cell divisions, we need to understand the biochemical activities of the contributing proteins. Here, we present a protocol to investigate how the ring-shaped condensin complex sequentially and topologically entraps two DNA substrates. We describe the steps to prepare purified Schizosaccharomyces pombe condensin, as well as bulk biochemical assays to monitor the first and second DNA capture reactions. This protocol may facilitate further investigations of these essential genome organizers. For complete details on the use and execution of this protocol, please refer to Tang et al.1.

Establishment of dsDNA-dsDNA interactions by the condensin complex.

Condensin is a structural maintenance of chromosomes (SMC) complex family member thought to build mitotic chromosomes by DNA loop extrusion. However, condensin variants unable to extrude loops, yet proficient in chromosome formation, were recently described. Here, we explore how condensin might alternatively build chromosomes. Using bulk biochemical and single-molecule experiments with purified fission yeast condensin, we observe that individual condensins sequentially and topologically entrap two double-stranded DNAs (dsDNAs). Condensin loading transitions through a state requiring DNA bending, as proposed for the related cohesin complex. While cohesin then favors the capture of a second single-stranded DNA (ssDNA), second dsDNA capture emerges as a defining feature of condensin. We provide complementary in vivo evidence for DNA-DNA capture in the form of condensin-dependent chromatin contacts within, as well as between, chromosomes. Our results support a "diffusion capture" model in which condensin acts in mitotic chromosome formation by sequential dsDNA-dsDNA capture.

A Brownian ratchet model for DNA loop extrusion by the cohesin complex.

The cohesin complex topologically encircles DNA to promote sister chromatid cohesion. Alternatively, cohesin extrudes DNA loops, thought to reflect chromatin domain formation. Here, we propose a structure-based model explaining both activities. ATP and DNA binding promote cohesin conformational changes that guide DNA through a kleisin N-gate into a DNA gripping state. Two HEAT-repeat DNA binding modules, associated with cohesin's heads and hinge, are now juxtaposed. Gripping state disassembly, following ATP hydrolysis, triggers unidirectional hinge module movement, which completes topological DNA entry by directing DNA through the ATPase head gate. If head gate passage fails, hinge module motion creates a Brownian ratchet that, instead, drives loop extrusion. Molecular-mechanical simulations of gripping state formation and resolution cycles recapitulate experimentally observed DNA loop extrusion characteristics. Our model extends to asymmetric and symmetric loop extrusion, as well as z-loop formation. Loop extrusion by biased Brownian motion has important implications for chromosomal cohesin function.

When a cell divides, it has to ensure that each of its daughter cells inherits one copy of its genetic information. It does this by duplicating its chromosomes (the DNA molecules that encode the genome) and distributing one copy of each to its daughter cells. Once a cell duplicates a chromosome, the two identical chromosomes must be held together until the cell is ready to divide in two. A ring-shaped protein complex called cohesin does this by encircling the two chromosomes. Cohesin embraces both chromosome copies, as they emerge from the DNA replicating machinery. The complex is formed of several proteins that bind to a small molecule called ATP, whose arrival and subsequent breakdown release energy. Cohesin also interacts with DNA in a different way: it can create loops of chromatin (the complex formed by DNA and its packaging proteins) that help regulate the activity of genes. Experiments performed on single molecules isolated in the laboratory show that cohesin can form a small loop of DNA that is then enlarged through a process called DNA loop extrusion. However, it is not known whether loop extrusion occurs in the cell. Although both of cohesin's roles have to do with how DNA is organised in the cell, it remains unclear how a single protein complex can engage in two such different activities. To answer this question, Higashi et al. used a structure of cohesin from yeast cells gripping onto DNA to build a model that simulates how the complex interacts with chromosomes and chromatin. This model suggested that when ATP is broken down, the cohesin structure shifts and DNA enters the ring, allowing DNA to be entrapped and chromosomes to be bound together. However, a small change in how DNA is gripped initially could prevent it from entering the ring, creating a ratchet mechanism that forms and enlarges a DNA loop. This molecular model helps explain how cohesin can either encircle DNA or create loops. However, Higashi et al.'s findings also raise the question of whether loop extrusion is possible inside cells, where DNA is densely packed and bound to proteins which could be obstacles to loop extrusion. Further research to engineer cohesin that can only perform one of these roles would help to clarify their individual contributions in the cell.

Dynamic architecture of the Escherichia coli structural maintenance of chromosomes (SMC) complex, MukBEF.

Ubiquitous Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organize and individualize chromosomes, thereby facilitating chromosome segregation. They utilize cycles of adenosine triphosphate (ATP) binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contact sites. By analysing changes in the architecture and stoichiometry of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis, we demonstrate directly the formation of dimer of MukBEF dimer complexes, dependent on dimeric MukF kleisin. Using truncated and full length MukB, in combination with MukEF, we show that engagement of the MukB ATPase heads on nucleotide binding directs the formation of dimers of heads-engaged dimer complexes. Complex formation requires functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively, and MukE, which organizes the complexes by stabilizing binding of MukB heads to MukF. In the absence of head engagement, a MukF dimer bound by MukE forms complexes containing only a dimer of MukB. Finally, we demonstrate that cells expressing MukBEF complexes in which MukF is monomeric are Muk-, with the complexes failing to associate with chromosomes.

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

Structural characterization of the Streptococcus pneumoniae carbohydrate substrate-binding protein SP0092.

Streptococcus pneumoniae is an opportunistic respiratory pathogen that remains a major cause of morbidity and mortality globally, with infants and the elderly at the highest risk. S. pneumoniae relies entirely on carbohydrates as a source of carbon and dedicates a third of all uptake systems to carbohydrate import. The structure of the carbohydrate-free substrate-binding protein SP0092 at 1.61 Šresolution reveals it to belong to the newly proposed subclass G of substrate-binding proteins, with a ligand-binding pocket that is large enough to accommodate complex oligosaccharides. SP0092 is a dimer in solution and the crystal structure reveals a domain-swapped dimer with the monomer subunits in a closed conformation but in the absence of carbohydrate ligand. This closed conformation may be induced by dimer formation and could be used as a mechanism to regulate carbohydrate uptake.