ABSTRACT
The cohesin ring, which is composed of the Smc1, Smc3, and Scc1 subunits, topologically embraces two sister chromatids from S phase until anaphase to ensure their precise segregation to the daughter cells. The opening of the ring is required for its loading on the chromosomes and unloading by the action of Wpl1 protein. Both loading and unloading are dependent on ATP hydrolysis by the Smc1 and Smc3 "head" domains, which engage to form two composite ATPase sites. Based on the available structures, we modeled the Saccharomyces cerevisiae Smc1/Smc3 head heterodimer and discovered that the Smc1/Smc3 interfaces at the two ATPase sites differ in the extent of protein contacts and stability after ATP hydrolysis. We identified smc1 and smc3 mutations that disrupt one of the interfaces and block the Wpl1-mediated release of cohesin from DNA. Thus, we provide structural insights into how the cohesin heads engage with each other.
Subject(s)
Acetyltransferases/genetics , Adenosine Triphosphate/chemistry , Cell Cycle Proteins/chemistry , Chromosomal Proteins, Non-Histone/chemistry , Nuclear Proteins/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Acetyltransferases/metabolism , Amino Acid Motifs , Binding Sites , Catalytic Domain , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromatids/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Dimerization , Hydrolysis , Models, Molecular , Mutation , Nuclear Proteins/metabolism , Protein Binding , Protein Multimerization , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , CohesinsABSTRACT
Cohesin is a ring-shaped protein complex that is capable of embracing DNA. Most of the ring circumference is comprised of the anti-parallel intramolecular coiled coils of the Smc1 and Smc3 proteins, which connect globular head and hinge domains. Smc coiled coil arms contain multiple acetylated and ubiquitylated lysines. To investigate the role of these modifications, we substituted lysines for arginines to mimic the unmodified state and uncovered genetic interaction between the Smc arms. Using scanning force microscopy, we show that wild-type Smc arms associate with each other when the complex is not on DNA. Deacetylation of the Smc1/Smc3 dimers promotes arms' dissociation. Smc arginine mutants display loose packing of the Smc arms and, although they dimerize at the hinges, fail to connect the heads and associate with the DNA. Our findings highlight the importance of a "collapsed ring," or "rod," conformation of cohesin for its loading on the chromosomes.
Subject(s)
Cell Cycle Proteins/chemistry , Chromosomal Proteins, Non-Histone/chemistry , DNA, Fungal/chemistry , Lysine/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/genetics , Acetylation , Amino Acid Substitution , Animals , Arginine/metabolism , Baculoviridae/genetics , Baculoviridae/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromatids/chemistry , Chromatids/metabolism , Chromatids/ultrastructure , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Fungal/chemistry , Chromosomes, Fungal/metabolism , Chromosomes, Fungal/ultrastructure , Cloning, Molecular , DNA, Fungal/genetics , DNA, Fungal/metabolism , Gene Expression , Gene Expression Regulation, Fungal , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sf9 Cells , Signal Transduction , Spodoptera , CohesinsABSTRACT
Extensively drug-resistant (XDR) Klebsiella pneumoniae is an emerging pathogen in Singapore. With limited therapeutic options available, combination antibiotics may be the only viable option. In this study, we aimed to elucidate effective antibiotic combinations against XDR K. pneumoniae isolates. Six NDM-1-producing and two OXA-181-producing K. pneumoniae strains were exposed to 12 antibiotics alone and in combination via time-kill studies. A hollow-fiber infection model (HFIM) with pharmacokinetic validation was used to simulate clinically relevant tigecycline-plus-meropenem dosing regimens against 2 XDR K. pneumoniae isolates over 240 h. The emergence of resistance against tigecycline was quantified using drug-free and selective (tigecycline at 3× the MIC) media. The in vitro growth rates were determined and serial passages on drug-free and selective media were carried out on resistant isolates obtained at 240 h. Both the polymyxin B and tigecycline MICs ranged from 1 to 4 mg/liter. In single time-kill studies, all antibiotics alone demonstrated regrowth at 24 h, except for polymyxin B against 2 isolates. Tigecycline plus meropenem was found to be bactericidal in 50% of the isolates. For the isolates that produced OXA-181-like carbapenemases, none of the 55 tested antibiotic combinations was bactericidal. Against 2 isolates in the HFIM, tigecycline plus meropenem achieved a >90% reduction in bacterial burden for 96 h before regrowth was observed until 10(9) CFU/ml at 240 h. Phenotypically stable and resistant isolates, which were recovered from tigecycline-supplemented plates post-HFIM studies, had lower growth rates than those of their respective parent isolates, possibly implying a substantial biofitness deficit in this population. We found that tigecycline plus meropenem may be a potential antibiotic combination for XDR K. pneumoniae infections, but its efficacy was strain specific.
