Structural Maintenance of Chromosomes Protein C-terminal Domains Bind Preferentially to DNA with Secondary Structure

Alexandre T Akhmedov,Christian Frei,Monika Tsai-Pflugfelder,Börries Kemper,Susan M Gasser,Rolf Jessberger

doi:10.1074/jbc.273.37.24088

Abstract

Structural maintenance of chromosomes (SMC) proteins interact with DNA in chromosome condensation, sister chromatid cohesion, DNA recombination, and gene dosage compensation. How individual SMC proteins and their functional domains bind DNA has not been described. We demonstrate the ability of the C-terminal domains of Saccharomyces cerevisiae SMC1 and SMC2 proteins, representing two major subfamilies with different functions, to bind DNA in an ATP-independent manner. Three levels of DNA binding specificity were observed: 1) a >100-fold preference for double-stranded versus single-stranded DNA; 2) a high affinity for DNA fragments able to form secondary structures and for synthetic cruciform DNA molecules; and 3) a strong preference for AT-rich DNA fragments of particular types. These include fragments from the scaffold-associated regions, and an alternating poly(dA-dT)-poly(dT-dA) synthetic polymer, as opposed to a variety of other polymers. Reannealing of complementary DNA strands is also promoted primarily by the C-terminal domains. Consistent with their in vitro DNA binding activity, we show that overexpression of the SMC C termini increases plasmid loss without altering viability or cell cycle progression.

Highlights

Structural maintenance of chromosomes (SMC) proteins interact with DNA in chromosome condensation, sister chromatid cohesion, DNA recombination, and gene dosage compensation
Examples are the X. laevis 13 S condesin, in which XCAP-C and -E (SMC4 and 2, respectively) are complexed with three non-structural maintenance of chromosomes (SMC) proteins, required for chromosome condensation [12]; the bovine recombination protein complex RC-1, in which SMC1 and SMC3 molecules are complexed with DNA ligase III and DNA polymerase ⑀ [19]; or the C. elegans dosage compensation complex consisting of the two SMC proteins DPY-26 and MIX-1, associated with other proteins [16]
Purification of SMC Protein Terminal Domains—In order to analyze the biochemical properties of the individual SMC proteins, the N- and C-terminal globular domains of the S. cerevisiae SMC1 and SMC2 proteins were expressed in bacteria as GST-fusion proteins and were purified in their native forms

Summary

EXPERIMENTAL PROCEDURES

Purification of S. cerevisiae SMC Protein Domains—N- and C-terminal domains of SMC1 and SMC2 were expressed in E. coli as GST fusion proteins. Gel Retardation Assays—These assays were performed in 10-␮l reaction mixtures containing 0.5–1 ng (3000 – 6000 cpm) of 32P 5Ј-labeled DNA in 20 mM HEPES (pH 7.5), 1 mM dithiothreitol, 100 ␮g/ml BSA, and protein as indicated. Transformed cells were grown on selective plates (minimal medium, lacking adenine and uracil) containing glucose to repress SMC expression. To analyze the influence of overexpressed SMC on pASZ11 plasmid loss, transformed yeast cells were either allowed to grow directly in synthetic drop-out medium lacking uracil and supplemented with adenine (20 mg/liter), 2% (w/v) galactose, 3% (v/v) glycerol, and 2% (v/v) lactate for 6 – 8 generations, or after depleting glucose by preincubating the cells for 12 h in synthetic dropout medium lacking uracil and adenine and supplemented with 3% (v/v) glycerol, and 2% (v/v) lactate. Control cultures were treated identically, but growth was continued on glucose throughout

RESULTS

SMC Proteins Bind to DNA with Secondary Structure

No protein

TABLE II Quantitation of reannealing product

Plasmid loss per generation

DISCUSSION