Biochemical characterization of the Structural Maintenance of Chromosomes (SMC) complex from Bacillus subtilis

Structural Maintenance of Chromosomes (SMC) proteins play a key role in the chromosome dynamics throughout the cell cycle in almost all species from bacteria to eukaryotes. Proteins from SMC family are involved in a number of processes, such as chromosomes condensation and segregation, sister-chromatid cohesion and DNA double strand break repair. All SMC proteins share a typical structure and consist of N- and C-terminal domains carrying the ATPase motif, the hinge domain and two central coiled-coil domains. Terminal domains come together to form one head domain, while coiled coil domains form a single coiled coil. SMC proteins form an intermolecular dimer via interaction of hinge domains. All eukaryotic SMCs perform their function in complex with a number of other none SMC subunits and recently, two novel prokaryotic proteins, ScpA and ScpB, have been found to interact with bacterial SMC in vivo. In this work, biochemical studies were performed to understand the properties of B. subtilis SMC, ScpA and ScpB in vitro, and to elucidate the mechanism of their action in vivo. The main state of ScpB in solution was found to be a dimer, while ScpA exists in both monomeric and dimeric forms. Using different approaches, such as size exclusion chromatography, gel shift assay and sucrose gradient ultracentrifugation, I found that SMC, ScpA and ScpB indeed form a ternary complex, which most likely consists of one SMC dimer, two ScpAs and two ScpB dimers. ScpA and ScpB were also able to form two types of complexes in absence of SMC: one formed by one ScpA and a dimer of ScpB, and a larger complex most likely consisting of two ScpAs and two ScpB dimers. ScpB was shown to interact with SMC indirectly only in presence of ScpA, and ScpA interacted stably with the SMC head domains only in the presence of ScpB. In addition, gel filtration assays suggested that the SMC complex is most likely formed by direct binding of the ScpA/ScpB complex to SMC, rather than through binding of individual ScpA and ScpB molecules to SMC. Sucrose gradient analysis also showed that ScpA, ScpB and SMC are present as a complex as well as in non-complexed form, indicating that the SMC complex is in a dynamic state in vivo. Another aspect investigated here were the DNA binding properties of SMC, ScpA, ScpB as well as of different domains of SMC. I found that neither ScpA, nor ScpB are required for binding of SMC to DNA, and that they have no affinity to DNA in absence of SMC. Isolated hinge and head domains of SMC were also unable to bind DNA, thus, the complete SMC molecule is needed for proper function. SMC bound to dsDNA in a sequence independent manner, and based on data obtained from surface plasmon resonance experiments, binding to DNA occurred via formation of a closed ring-like structure. The data suggest that SMC interacts with DNA via dimerization of its head domains leading to the formation of a ring-like structure with DNA trapped in between the coiled-coil (domains) arms of SMC. Collaborative AFM studies have also shown ring formation by SMC, and large complex structures formed by SMCs were detected in solution that could explain why SMCs in bacterial cells are concentrated in certain regions of the cells (foci) and are not distributed (throughout the inner cellular space) all over the chromosome. Mutagenesis studies were another part of the project. SMC proteins have a weak ATPase activity and head domains contain conserved motifs that are typical for ABC-type ATPases. In this work I have shown, that ATP binding, but not ATP hydrolysis, is required for DNA binding of SMC. Additionally, none of these activities were required for complex formation with ScpA and ScpB, although formation of the SMC complex was less efficient in the mutant proteins. A model is suggested that ATP binding induces dimerization of head domains causing formation of a ring by SMC with DNA locked in the middle. Data obtained from gel filtration studies suggest that ScpA, in absence of ScpB, causes DNA release from SMC, while in the presence of ScpB, all three proteins form a stable complex. Therefore the condensation state of chromosomes in vivo could possibly be controlled by the levels of ScpB in the cell

Biochemical characterization of the Structural Maintenance of Chromosomes (SMC) complex from Bacillus subtilis

Structural Maintenance of Chromosomes (SMC) proteins play a key role in the chromosome dynamics throughout the cell cycle in almost all species from bacteria to eukaryotes. Proteins from SMC family are involved in a number of processes, such as chromosomes condensation and segregation, sister-chromatid cohesion and DNA double strand break repair. All SMC proteins share a typical structure and consist of N- and C-terminal domains carrying the ATPase motif, the hinge domain and two central coiled-coil domains. Terminal domains come together to form one head domain, while coiled coil domains form a single coiled coil. SMC proteins form an intermolecular dimer via interaction of hinge domains. All eukaryotic SMCs perform their function in complex with a number of other none SMC subunits and recently, two novel prokaryotic proteins, ScpA and ScpB, have been found to interact with bacterial SMC in vivo. In this work, biochemical studies were performed to understand the properties of B. subtilis SMC, ScpA and ScpB in vitro, and to elucidate the mechanism of their action in vivo. The main state of ScpB in solution was found to be a dimer, while ScpA exists in both monomeric and dimeric forms. Using different approaches, such as size exclusion chromatography, gel shift assay and sucrose gradient ultracentrifugation, I found that SMC, ScpA and ScpB indeed form a ternary complex, which most likely consists of one SMC dimer, two ScpAs and two ScpB dimers. ScpA and ScpB were also able to form two types of complexes in absence of SMC: one formed by one ScpA and a dimer of ScpB, and a larger complex most likely consisting of two ScpAs and two ScpB dimers. ScpB was shown to interact with SMC indirectly only in presence of ScpA, and ScpA interacted stably with the SMC head domains only in the presence of ScpB. In addition, gel filtration assays suggested that the SMC complex is most likely formed by direct binding of the ScpA/ScpB complex to SMC, rather than through binding of individual ScpA and ScpB molecules to SMC. Sucrose gradient analysis also showed that ScpA, ScpB and SMC are present as a complex as well as in non-complexed form, indicating that the SMC complex is in a dynamic state in vivo. Another aspect investigated here were the DNA binding properties of SMC, ScpA, ScpB as well as of different domains of SMC. I found that neither ScpA, nor ScpB are required for binding of SMC to DNA, and that they have no affinity to DNA in absence of SMC. Isolated hinge and head domains of SMC were also unable to bind DNA, thus, the complete SMC molecule is needed for proper function. SMC bound to dsDNA in a sequence independent manner, and based on data obtained from surface plasmon resonance experiments, binding to DNA occurred via formation of a closed ring-like structure. The data suggest that SMC interacts with DNA via dimerization of its head domains leading to the formation of a ring-like structure with DNA trapped in between the coiled-coil (domains) arms of SMC. Collaborative AFM studies have also shown ring formation by SMC, and large complex structures formed by SMCs were detected in solution that could explain why SMCs in bacterial cells are concentrated in certain regions of the cells (foci) and are not distributed (throughout the inner cellular space) all over the chromosome. Mutagenesis studies were another part of the project. SMC proteins have a weak ATPase activity and head domains contain conserved motifs that are typical for ABC-type ATPases. In this work I have shown, that ATP binding, but not ATP hydrolysis, is required for DNA binding of SMC. Additionally, none of these activities were required for complex formation with ScpA and ScpB, although formation of the SMC complex was less efficient in the mutant proteins. A model is suggested that ATP binding induces dimerization of head domains causing formation of a ring by SMC with DNA locked in the middle. Data obtained from gel filtration studies suggest that ScpA, in absence of ScpB, causes DNA release from SMC, while in the presence of ScpB, all three proteins form a stable complex. Therefore the condensation state of chromosomes in vivo could possibly be controlled by the levels of ScpB in the cell

Dynamic assembly, localization and proteolysis of the Bacillus subtilis SMC complex

BACKGROUND: SMC proteins are key components of several protein complexes that perform vital tasks in different chromosome dynamics. Bacterial SMC forms a complex with ScpA and ScpB that is essential for chromosome arrangement and segregation. The complex localizes to discrete centres on the nucleoids that during most of the time of the cell cycle localize in a bipolar manner. The complex binds to DNA and condenses DNA in an as yet unknown manner. RESULTS: We show that in vitro, ScpA and ScpB form different complexes with each other, among which the level of the putative 2 ScpA/4 ScpB complex showed a pronounced decrease in level upon addition of SMC protein. Different mutations of the ATPase-binding pocket of SMC reduced, but did not abolish interaction of mutant SMC with ScpA and ScpB. The loss of SMC ATPase activity led to a loss of function in vivo, and abolished proper localization of the SMC complex. The formation of bipolar SMC centres was also lost after repression of gyrase activity, and was abnormal during inhibition of replication, resulting in single central clusters. Resumption of replication quickly re-established bipolar SMC centres, showing that proper localization depends on ongoing replication. We also found that the SMC protein is subject to induced proteolysis, most strikingly as cells enter stationary phase, which is partly achieved by ClpX and LonA proteases. Atomic force microscopy revealed the existence of high order rosette-like SMC structures in vitro, which might explain the formation of the SMC centres in vivo. CONCLUSION: Our data suggest that a ScpA/ScpB sub-complex is directly recruited into the SMC complex. This process does not require SMC ATPase activity, which, however, appears to facilitate loading of ScpA and ScpB. Thus, the activity of SMC could be regulated through binding and release of ScpA and ScpB, which has been shown to affect SMC ATPase activity. The proper bipolar localization of the SMC complex depends on a variety of physiological aspects: ongoing replication, ATPase activity and chromosome supercoiling. Because the cellular concentration of SMC protein is also regulated at the posttranscriptional level, the activity of SMC is apparently regulated at multiple levels

C-Terminal regions of topoisomerase IIα and IIβ determine isoform-specific functioning of the enzymes in vivo

Topoisomerase II removes supercoils and catenanes generated during DNA metabolic processes such as transcription and replication. Vertebrate cells express two genetically distinct isoforms (α and β) with similar structures and biochemical activities but different biological roles. Topoisomerase IIα is essential for cell proliferation, whereas topoisomerase IIβ is required only for aspects of nerve growth and brain development. To identify the structural features responsible for these differences, we exchanged the divergent C-terminal regions (CTRs) of the two human isoforms (α 1173-1531 and β 1186-1621) and tested the resulting hybrids for complementation of a conditional topoisomerase IIα knockout in human cells. Proliferation was fully supported by all enzymes bearing the α CTR. The α CTR also promoted chromosome binding of both enzyme cores, and was by itself chromosome-bound, suggesting a role in enzyme targeting during mitosis. In contrast, enzymes bearing the β CTR supported proliferation only rarely and when expressed at unusually high levels. A similar analysis of the divergent N-terminal regions (α 1-27 and β 1-43) revealed no role in isoform-specific functions. Our results show that it is the CTRs of human topoisomerase II that determine their isoform-specific functions in proliferating cells. They also indicate persistence of some functional redundancy between the two isoforms

UVA-induced carbon-centered radicals in lightly pigmented cells detected using ESR spectroscopy

Ultraviolet-A and melanin are implicated in melanoma, but whether melanin in vivo screens or acts as a UVA photosensitiser is debated. Here, we investigate the effect of UVA-irradiation on non-pigmented, lightly and darkly pigmented melanocytes and melanoma cells using electron spin resonance (ESR) spectroscopy. Using the spin trap 5,5 Dimethyl-1-pyrroline N-oxide (DMPO), carbon adducts were detected in all cells. However, higher levels of carbon adducts were detected in lightly pigmented cells than in non-pigmented or darkly pigmented cells. Nevertheless, when melanin levels were artificially increased in lightly pigmented cells by incubation with L-Tyrosine, the levels of carbon adducts decreased significantly. Carbon adducts were also detected in UVA-irradiated melanin-free cell nuclei, DNA-melanin systems, and the nucleoside 2’-deoxyguanosine combined with melanin, whereas they were only weakly detected in irradiated synthetic melanin and not at all in irradiated 2’-deoxyguanosine. The similarity of these carbon adducts suggests they may be derived from nucleic acid– guanine – radicals. These observations suggest that melanin is not consistently a UVA screen against free-radical formation in pigmented cells, but may also act as a photosensitizer for the formation of nucleic acid radicals in addition to superoxide. The findings are important for our understanding of the mechanism of damage caused by the UVA component of sunlight in non-melanoma and melanoma cells, and hence the causes of skin cancer

Measurement of t(t)over-bar normalised multi-differential cross sections in pp collisions at root s=13 TeV, and simultaneous determination of the strong coupling strength, top quark pole mass, and parton distribution functions

Peer reviewe

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe

Measurement of prompt open-charm production cross sections in proton-proton collisions at root s=13 TeV

The production cross sections for prompt open-charm mesons in proton-proton collisions at a center-of-mass energy of 13TeV are reported. The measurement is performed using a data sample collected by the CMS experiment corresponding to an integrated luminosity of 29 nb(-1). The differential production cross sections of the D*(+/-), D-+/-, and D-0 ((D) over bar (0)) mesons are presented in ranges of transverse momentum and pseudorapidity 4 < p(T) < 100 GeV and vertical bar eta vertical bar < 2.1, respectively. The results are compared to several theoretical calculations and to previous measurements.Peer reviewe

Measurement of the top quark mass using events with a single reconstructed top quark in pp collisions at root s=13 TeV

Abstract:A measurement of the top quark mass is performed using a data sample en-riched with single top quark events produced in thetchannel. The study is based on proton-proton collision data, corresponding to an integrated luminosity of 35.9 fb−1, recorded at√s= 13TeV by the CMS experiment at the LHC in 2016. Candidate events are selectedby requiring an isolated high-momentum lepton (muon or electron) and exactly two jets,of which one is identified as originating from a bottom quark. Multivariate discriminantsare designed to separate the signal from the background. Optimized thresholds are placedon the discriminant outputs to obtain an event sample with high signal purity. The topquark mass is found to be172.13+0.76−0.77GeV, where the uncertainty includes both the sta-tistical and systematic components, reaching sub-GeV precision for the first time in thisevent topology. The masses of the top quark and antiquark are also determined separatelyusing the lepton charge in the final state, from which the mass ratio and difference aredetermined to be0.9952+0.0079−0.0104and0.83+1.79−1.35GeV, respectively. The results are consistentwithCPTinvariance

Search for a heavy resonance decaying to a top quark and a w boson at √s = 13 tev in the fully hadronic final state

A search for a heavy resonance decaying to a top quark and a W boson in the fully hadronic final state is presented. The analysis is performed using data from proton-proton collisions at a center-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 137 fb−1 recorded by the CMS experiment at the LHC. The search is focused on heavy resonances, where the decay products of each top quark or W boson are expected to be reconstructed as a single, large-radius jet with a distinct substructure. The production of an excited bottom quark, b*, is used as a benchmark when setting limits on the cross section for a heavy resonance decaying to a top quark and a W boson. The hypotheses of b* quarks with left-handed, right-handed, and vector-like chiralities are excluded at 95% confidence level for masses below 2.6, 2.8, and 3.1 TeV, respectively. These are the most stringent limits on the b* quark mass to date, extending the previous best limits by almost a factor of two