The Smc5/6 Core Complex Is a Structure-Specific DNA Binding and Compacting Machine.
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ABSTRACT:
SUBMITTER: Serrano D
PROVIDER: S-EPMC8224240 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also functions as a restriction factor preventing viral replication. Here, we report the cryogenic EM (cryo-EM) structure of the six-subunit budding yeast Smc5/6 holo-complex, reconstituted from recombinant proteins expressed in insect cells - providing both an architectural overview of the entire complex and an understanding of how the Nse1/3/4 subcomplex binds to the hetero-dimeric SMC protein core. In addition, we demonstrate that a region within the head domain of Smc5, equivalent to the 'W-loop' of Smc4 or 'F-loop' of Smc1, mediates an important interaction with Nse1. Notably, mutations that alter the surface-charge profile of the region of Nse1 which accepts the Smc5-loop, lead to a slow-growth phenotype and a global reduction in the chromatin-associated fraction of the Smc5/6 complex, as judged by single molecule localisation microscopy experiments in live yeast. Moreover, when taken together, our data indicates functional equivalence between the structurally unrelated KITE and HAWK accessory subunits associated with SMC complexes.
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Project description:Smc5/6 is an evolutionarily conserved SMC complex with roles in DNA replication and repair, as well as in viral DNA restriction. Understanding its multiple functions has been hampered by a lack of mechanistic studies on how the Smc5/6 complex associates with different types of DNA. Here we address this question by simultaneously visualizing the behavior of Smc5/6 on three types of DNA, namely double-stranded (ds) DNA, single-stranded (ss) DNA, and junction DNA formed by juxtaposed ss- and dsDNA, using correlative single-molecule fluorescence and force microscopy. We find that Smc5/6 displays distinct behaviors toward different types of DNA, dynamically associating with dsDNA while stably binding to junction DNA. Mechanistically, both the Nse1-3-4 subcomplex and ATP binding enhance the complex's dsDNA association. In contrast, Smc5/6's assembly onto ssDNA emanating from junction DNA, which occurs even in the presence high-affinity ssDNA binders, is aided by Nse1-3-4, but not by ATP. Moreover, we show that Smc5/6 protects junction DNA stability by preventing ssDNA annealing. The multifaceted DNA association behaviors of Smc5/6 provide a framework for understanding its diverse functions in genome maintenance and viral DNA restriction.
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Project description:SMC5/6 is a highly conserved protein complex related to cohesin and condensin, which are the key components of higher-order chromatin structures. The SMC5/6 complex is essential for proliferation in yeast and is involved in replication fork stability and processing. However, the precise mechanism of action of SMC5/6 is not known. Here we present evidence that the NSE1/NSE3/NSE4 sub-complex of SMC5/6 binds to double-stranded DNA without any preference for DNA-replication/recombination intermediates. Mutations of key basic residues within the NSE1/NSE3/NSE4 DNA-binding surface reduce binding to DNA in vitro. Their introduction into the Schizosaccharomyces pombe genome results in cell death or hypersensitivity to DNA damaging agents. Chromatin immunoprecipitation analysis of the hypomorphic nse3 DNA-binding mutant shows a reduced association of fission yeast SMC5/6 with chromatin. Based on our results, we propose a model for loading of the SMC5/6 complex onto the chromatin.
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Project description:Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction.
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Project description:The detection of DNA damage activates DNA repair pathways and checkpoints to allow time for repair. Ultimately, these responses must be coordinated to ensure that cell cycle progression is halted until repair is completed. Several multiprotein complexes containing members of the structural maintenance of chromosomes family of proteins have been described, including the condensin and cohesin complexes, that are critical for chromosomal organization. Here we show that the Smc5/Smc6 (Smc5/6) complex is required for a coordinated response to DNA damage and normal chromosome integrity. Fission yeast cells lacking functional Smc6 initiate a normal checkpoint response to DNA damage, culminating in the phosphorylation and activation of the Chk1 protein kinase. Despite this, cells enter a lethal mitosis, presumably without completion of DNA repair. Another subunit of the complex, Nse1, is a conserved member of this complex and is also required for this response. We propose that the failure to maintain a checkpoint response stems from the lack of ongoing DNA repair or from defective chromosomal organization, which is the signal to maintain a checkpoint arrest. The Smc5/6 complex is fundamental to genome integrity and may function with the condensin and cohesin complexes in a coordinated manner.
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Project description:DNA polymerase epsilon (Pol ?) is critical for genome duplication, but little is known about how post-translational modification regulates its function. Here we report that the Pol ? catalytic subunit Pol2 in yeast is sumoylated at a single lysine within a catalytic domain insertion uniquely possessed by Pol2 family members. We found that Pol2 sumoylation occurs specifically in S phase and is increased under conditions of replication fork blockade. Analyses of the genetic requirements of this modification indicate that Pol2 sumoylation is associated with replication fork progression and dependent on the Smc5/6 SUMO ligase known to promote DNA synthesis. Consistently, the pol2 sumoylation mutant phenotype suggests impaired replication progression and increased levels of gross chromosomal rearrangements. Our findings thus indicate a direct role for SUMO in Pol2-mediated DNA synthesis and a molecular basis for Smc5/6-mediated regulation of genome stability.
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Project description:The DNA double-strand breaks that initiate meiotic recombination are formed by topoisomerase relative Spo11, supported by conserved auxiliary factors. Because high-resolution structural data are lacking, many questions remain about the architecture of Spo11 and its partners and how they engage with DNA. We report cryo-EM structures at up to 3.3 Å resolution of DNA-bound core complexes of Saccharomyces cerevisiae Spo11 with Rec102, Rec104, and Ski8. In these structures, monomeric core complexes make extensive contacts with the DNA backbone and with the recessed 3'-OH and first 5' overhanging nucleotide, definitively establishing the molecular determinants of DNA end-binding specificity and providing insight into DNA cleavage preferences in vivo. The structures of individual subunits and their interfaces, supported by functional data in yeast, provide insight into the role of metal ions in DNA binding and uncover unexpected structural variation in homologs of the Top6BL component of the core complex.
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Project description:Hfq regulates bacterial gene expression post-transcriptionally by binding small RNAs and their target mRNAs, facilitating sRNA-mRNA annealing, typically resulting in translation inhibition and RNA turnover. Hfq is also found in the nucleoid and binds double-stranded (ds) DNA with a slight preference for A-tracts. Here, we present the crystal structure of the Escherichia coli Hfq Core bound to a 30 bp DNA, containing three 6 bp A-tracts. Although previously postulated to bind to the 'distal' face, three statistically disordered double stranded DNA molecules bind across the proximal face of the Hfq hexamer as parallel, straight rods with B-DNA like conformational properties. One DNA duplex spans the diameter of the hexamer and passes over the uridine-binding proximal-face pore, whereas the remaining DNA duplexes interact with the rims and serve as bridges between adjacent hexamers. Binding is sequence-independent with residues N13, R16, R17 and Q41 interacting exclusively with the DNA backbone. Atomic force microscopy data support the sequence-independent nature of the Hfq-DNA interaction and a role for Hfq in DNA compaction and nucleoid architecture. Our structure and nucleic acid-binding studies also provide insight into the mechanism of sequence-independent binding of Hfq to dsRNA stems, a function that is critical for proper riboregulation.
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Project description:Eukaryotic cells employ three SMC (structural maintenance of chromosomes) complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo-complex. We found that the Nse5/6 sub-complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross-linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross-linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non-productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.
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