Condensin, an SMC (structural maintenance of chromosomes) protein complex, extrudes DNA loops using an ATP‐dependent mechanism that remains to be elucidated. Here, we show how condensin activity ...alters the topology of the interacting DNA. High condensin concentrations restrain positive DNA supercoils. However, in experimental conditions of DNA loop extrusion, condensin restrains negative supercoils. Namely, following ATP‐mediated loading onto DNA, each condensin complex constrains a DNA linking number difference (∆Lk) of −0.4. This ∆Lk increases to −0.8 during ATP binding and resets to −0.4 upon ATP hydrolysis. These changes in DNA topology do not involve DNA unwinding, do not spread outside the condensin‐DNA complex and can occur in the absence of the condensin subunit Ycg1. These findings indicate that during ATP binding, a short DNA domain delimited by condensin is pinched into a negatively supercoiled loop. We propose that this loop is the feeding segment of DNA that is subsequently merged to enlarge an extruding loop. Such a “pinch and merge” mechanism implies that two DNA‐binding sites produce the feeding loop, while a third site, plausibly involving Ycg1, might anchor the extruding loop.
Synopsis
How the structural maintenance of chromosomes (SMC) complex condensin extrudes DNA loops via ATPase activity remains unclear. Results showing that yeast condensin activity produces cyclic alterations in the topology of its interacting DNA suggest how it generates DNA translocation steps to achieve loop extrusion.
Condensin activity restrains negative DNA supercoils upon ATP usage.
The Ycg1 subunit is not required to restrain negative DNA supercoils.
The ATP‐bound condensin pinches a short left‐handed loop of DNA.
The transiently constrained DNA loop is not the extruding loop.
A “pinch and merge” mechanism of DNA loop extrusion is proposed.
Condensin activity produces cyclic alterations in the topology of its interacting DNA, suggesting how this SMC complex generates DNA translocation steps to extrude DNA loops.
The dominant model for eukaryotic cell cycle control proposes that cell cycle progression is driven by a succession of CDK complexes with different substrate specificities. However, in fission yeast ...it has been shown that a single CDK complex generated by the fusion of the Cdc13 cyclin with the CDK protein Cdc2 can drive the mitotic cell cycle. Meiosis is a modified cell cycle programme in which a single S-phase is followed by two consecutive rounds of chromosome segregation. Here we systematically analyse the requirements of the different fission yeast cyclins for meiotic cell cycle progression. We also show that a single Cdc13-Cdc2 complex, in the absence of the other cyclins, can drive the meiotic cell cycle. We propose that qualitatively different CDK complexes are not absolutely required for cell cycle progression either during mitosis or meiosis, and that a single CDK complex can drive both cell cycle programmes.
SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many ...molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6’s interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex’s enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.
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•The Smc5/6 holocomplex can form oligomers and translocate on double-stranded DNA•The complex can simultaneously bind single-stranded and double-stranded DNA•Smc5/6 is recruited to ssDNA-dsDNA junction structures mimicking replication forks•The holocomplex indirectly stabilizes the ssDNA-binding protein RPA on ssDNA
Tanasie et al. combine single-molecule microscopy and force manipulation to investigate the interaction of the Smc5/6 holocomplexes with fork junction structures mimicking DNA replication and repair intermediates. They show that Smc5/6 targets and stabilizes these DNA substrates, suggesting a model for the Smc5/6-mediated recognition of replication stress or DNA lesions.
Abstract
The role of Rad53 in response to a DNA lesion is central for the accurate orchestration of the DNA damage response. Rad53 activation relies on its phosphorylation by Mec1 and its own ...autophosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 activation has been well documented, less is known about the processes that counteract its activity along the repair of a DNA adduct. Here, we describe that PP4 phosphatase is required to avoid Rad53 hyper-phosphorylation during the repair of a double-strand break, a process that impacts on the phosphorylation status of multiple factors involved in the DNA damage response. PP4-dependent Rad53 dephosphorylation stimulates DNA end resection by relieving the negative effect that Rad9 exerts over the Sgs1/Dna2 exonuclease complex. Consequently, elimination of PP4 activity affects resection and repair by single-strand annealing, defects that are bypassed by reducing Rad53 hyperphosphorylation. These results confirm that Rad53 phosphorylation is controlled by PP4 during the repair of a DNA lesion and demonstrate that the attenuation of its kinase activity during the initial steps of the repair process is essential to efficiently enhance recombinational DNA repair pathways that depend on long-range resection for their success.
Cohesin is a regulator of genome architecture with roles in sister chromatid cohesion and chromosome compaction. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes. ...Here, we show that the role of cohesin in chromosome organization requires the histone chaperone FACT ('facilitates chromatin transcription') in Saccharomyces cerevisiae. We find that FACT interacts directly with cohesin, and is dynamically required for its localization on chromatin. Depletion of FACT in metaphase cells prevents cohesin accumulation at pericentric regions and causes reduced binding on chromosome arms. Using the Hi-C technique, we show that cohesin-dependent TAD (topological associated domain)-like structures in G1 and metaphase chromosomes are reduced in the absence of FACT. Sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our data show that FACT contributes to the formation of cohesin-dependent TADs, thus uncovering a new role for this complex in nuclear organization during interphase and mitotic chromosome folding.
Replication of a damaged DNA template can threaten the integrity of the genome, requiring the use of various mechanisms to tolerate DNA lesions. The Smc5/6 complex, together with the Nse2/Mms21 SUMO ...ligase, plays essential roles in genome stability through undefined tasks at damaged replication forks. Various subunits within the Smc5/6 complex are substrates of Nse2, but we currently do not know the role of these modifications. Here we show that sumoylation of Smc5 is targeted to its coiled-coil domain, is upregulated by replication fork damage, and participates in bypass of DNA lesions. smc5-KR mutant cells display defects in formation of sister chromatid junctions and higher translesion synthesis. Also, we provide evidence indicating that Smc5 sumoylation modulates Mph1-dependent fork regression, acting synergistically with other pathways to promote chromosome disjunction. We propose that sumoylation of Smc5 enhances physical remodeling of damaged forks, avoiding the use of a more mutagenic tolerance pathway.
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•Smc5-SUMO is a specific read-out for damaged replication forks•SUMO preferentially targets lysines in the coiled-coil domain of Smc5•smc5-KR is epistatic to MPH1, upregulating TLS and reducing strand exchange•Defects in Smc5 sumoylation are normally backed up by the Mms4-Mus81 nuclease
Zapatka et al. show that sumoylation of Smc5 helps cells tolerate DNA lesions at damaged replication forks in an error-free mode. Using unsumoylatable smc5-KR mutants, they show that this modification operates through Mph1 in fork regression, working in parallel with several nucleases and helicases to promote chromosome segregation.
Complexes containing a pair of structural maintenance of chromosomes (SMC) family proteins are fundamental for the three-dimensional (3D) organization of genomes in all domains of life. The ...eukaryotic SMC complexes cohesin and condensin are thought to fold interphase and mitotic chromosomes, respectively, into large loop domains, although the underlying molecular mechanisms have remained unknown. We used cryo-EM to investigate the nucleotide-driven reaction cycle of condensin from the budding yeast Saccharomyces cerevisiae. Our structures of the five-subunit condensin holo complex at different functional stages suggest that ATP binding induces the transition of the SMC coiled coils from a folded-rod conformation into a more open architecture. ATP binding simultaneously triggers the exchange of the two HEAT-repeat subunits bound to the SMC ATPase head domains. We propose that these steps result in the interconversion of DNA-binding sites in the catalytic core of condensin, forming the basis of the DNA translocation and loop-extrusion activities.
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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•Purification of enzymatically active Smc5/6 yeast holocomplex•Smc5/6 coiled-coils exhibit a folded conformation•Smc5/6 stabilizes DNA plectonemes•Smc5/6 compacts DNA against low forces in an ATP-dependent manner
Gutierrez-Escribano et al. purify the entire Smc5/6 holocomplex, retaining full enzymatic function. The Smc5/6 complex compacts DNA against low force and stabilizes DNA crosses present in supercoiled and catenated DNA. These findings indicate that many Smc5/6 functions occur through stabilization of DNA tertiary structure.
Candida albicans is a major invasive fungal pathogen in humans. An important virulence factor is its ability to switch between the yeast and hyphal forms, and these filamentous forms are important in ...tissue penetration and invasion. A common feature for filamentous growth is the ability to inhibit cell separation after cytokinesis, although it is poorly understood how this process is regulated developmentally. In C. albicans, the formation of filaments during hyphal growth requires changes in septin ring dynamics. In this work, we studied the functional relationship between septins and the transcription factor Ace2, which controls the expression of enzymes that catalyze septum degradation. We found that alternative translation initiation produces two Ace2 isoforms. While full-length Ace2, Ace2L, influences septin dynamics in a transcription-independent manner in hyphal cells but not in yeast cells, the use of methionine-55 as the initiation codon gives rise to Ace2S, which functions as the nuclear transcription factor required for the expression of cell separation genes. Genetic evidence indicates that Ace2L influences the incorporation of the Sep7 septin to hyphal septin rings in order to avoid inappropriate activation of cell separation during filamentous growth. Interestingly, a natural single nucleotide polymorphism (SNP) present in the C. albicans WO-1 background and other C. albicans commensal and clinical isolates generates a stop codon in the ninth codon of Ace2L that mimics the phenotype of cells lacking Ace2L. Finally, we report that Ace2L and Ace2S interact with the NDR kinase Cbk1 and that impairing activity of this kinase results in a defect in septin dynamics similar to that of hyphal cells lacking Ace2L. Together, our findings identify Ace2L and the NDR kinase Cbk1 as new elements of the signaling system that modify septin ring dynamics in hyphae to allow cell-chain formation, a feature that appears to have evolved in specific C. albicans lineages.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In nature, many microorganisms form specialized complex, multicellular, surface-attached communities called biofilms. These communities play critical roles in microbial pathogenesis. The fungal ...pathogen Candida albicans is associated with catheter-based infections due to its ability to establish biofilms. The transcription factor Bcr1 is a master regulator of C. albicans biofilm development, although the full extent of its regulation remains unknown. Here, we report that Bcr1 is a phosphoprotein that physically interacts with the NDR kinase Cbk1 and undergoes Cbk1-dependent phosphorylation. Mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to alanine markedly impaired Bcr1 function during biofilm formation and virulence in a mouse model of disseminated candidiasis. Cells lacking Cbk1, or any of its upstream activators, also had reduced biofilm development. Notably, mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to glutamate in cbk1Δ cells upregulated the transcription of Bcr1-dependent genes and partially rescued the biofilm defects of a cbk1Δ strain. Therefore, our data uncovered a novel role of the NDR/LATS kinase Cbk1 in the regulation of biofilm development through the control of Bcr1.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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