Using DNA as a Fiducial Marker To Study SMC Complex Interactions with the Atomic Force Microscope

Abstract

Atomic force microscopy can potentially provide information on protein volumes, shapes, and interactions but is susceptible to variable tip-induced artifacts. In this study, we present an atomic force microscopy approach that can measure volumes of nonglobular polypeptides such as structural maintenance of chromosomes (SMC) proteins, and use it to study the interactions that occur within and between SMC complexes. Together with the protein of interest, we coadsorb a DNA molecule and use it as a fiducial marker to account for tip-induced artifacts that affect both protein and DNA, allowing normalization of protein volumes from images taken on different days and with different tips. This approach significantly reduced the error associated with volume analysis, and allowed determination of the oligomeric states and architecture of the Bacillus subtilis SMC complex, formed by the SMC protein, and by the smaller ScpA and ScpB subunits. This work reveals that SMC and ScpB are dimers and that ScpA is a stable monomer. Moreover, whereas ScpA binds directly to SMC, ScpB only binds to SMC in the presence of ScpA. Notably, the presence of both ScpA and ScpB favored the formation of higher-order structures of SMC complexes, suggesting a role for these subunits in the organization of SMC oligomers.

Single molecule analysis of DNA wrapping and looping by a circular 14mer wheel of the bacteriophage 186 CI repressor

“…The measured volume of the 186 CI particles in the presence of specific 186 DNA (632 ± 152 nm 3 ; n = 975) is consistent with the molecular weight of a 6His-tagged CI 14mer (22 590 × 14 = 316 kDa) (Supplementary Figure S1). Our calibration curve is remarkably similar to one recently published using a fiducial marker for precise volume calibration (17). …”

Structural Maintenance of Chromosome Complex in Bacteria

“…This results in an asymmetric, tripartite Smc-ScpAB ring, similar in shape and size to cohesin, which is most probably the functional arrangement of the closed complex [Bürmann et al, 2013]. Using AFM, the formation of higher-order structures of several interacting Smc dimers were observed, which had a rosette-like arrangement , or in another study were only seen in the presence of both ScpA and ScpB [Fuentes-Perez et al, 2012], indicating the possibility of Smc and of the SMC complex to form interactions between single SMC complexes (note that we are using 'Smc' when a specific SMC protein is meant, and 'SMC' when a protein from the family is meant). A key question to be answered in the future is how an ScpAB complex can bind to a symmetrical Smc dimer in an asymmetric manner: in principle, two ScpAB could bind to each Smc arm/head in an SMC dimer, which would prevent ring formation; how this is prevented at a molecular level is an intriguing problem to be solved.…”

“…Of note, the exact stoichiometry of the Smc/ScpA/ScpB complex has been quite controversial. Biochemical and AFM analysis revealed that the stoichiometry of the SMC complex is 2: 2:4 SMC:ScpA:ScpB [Fuentes-Perez et al, 2012;Mascarenhas et al, 2005], though also a stoichiometry of 1: 1:1 was proposed in another study [Hirano and Hirano, 2004]. However, as mentioned before, two recent studies proposed a 2: 1:2 stoichiometry of Smc-ScpAB based on the crystal structure, gel filtration and SDS page [Bür-mann et al, 2013;Kamada et al, 2013].…”

“…1 a). Electron microscopy and atomic force microscopy (AFM) studies revealed that the dimers change between different confirmations: an open V shape and a closed O shape [Fuentes-Perez et al, 2012;Melby et al, 1998]. The average length of these structures is 45 ± 6 nm width and the height is 0.7 ± 1 Å [Fuentes-Perez et al, 2012].…”

The role of ATP-dependent machines in regulating genome topology

“…How multiple DNA segments might be coordinated by SMC action is not known at a physical level; however, a recent structure involving the SMC-like protein Rad50 demonstrates that at least one duplex can be bound by the ATPase domain directly (Figure 4d [109]). Single molecule work has recapitulated the SMC-medicated compaction of DNA [110–112], and AFM studies suggest that multiple SMCs may also interact with one another to promote long-range DNA compaction [113,114]. Recent structural and crosslinking studies have indicated that the coiled-coiled domains of SMC complexes can undergo a conformational rearrangement upon binding of ATP and DNA, which may foster communication between the hinge and ATPase domains [115], or possibly promote hinge opening [116,117]; for cohesin, proteolytic cleavage of one of the accessory subunits also regulates subunit opening and DNA release [118,119].…”