scholarly journals CryoEM structures of open dimers of Gyrase A in complex with DNA illuminate mechanism of strand passage

2018 ◽  
Author(s):  
Katarzyna M. Soczek ◽  
Tim Grant ◽  
Peter B. Rosenthal ◽  
Alfonso Mondragon
Keyword(s):  
Conformational Changes ◽  
Dna Cleavage ◽  
Atp Hydrolysis ◽  
Unique Type ◽  
Dna Molecule ◽  
Gyrase A ◽  
A Subunit ◽  
Important Intermediate ◽  
Dna Strand ◽  
Strand Passage

AbstractGyrase is a unique type IIA topoisomerase that uses ATP hydrolysis to maintain the negatively supercoiled state of bacterial DNA. In order to perform its function, gyrase undergoes a sequence of conformational changes that consist of concerted gate openings, DNA cleavage, and DNA strand passage events. Structures where the transported DNA molecule (T-segment) is trapped by the A subunit have not been observed. Here we present the cryoEM structures of two oligomeric complexes of open gyrase A dimers and DNA. The protein subunits in these complexes were solved to 4 Å and 5.16 Å resolution. One of the complexes traps a linear DNA molecule, a putative T-segment, which interacts with the open gyrase A dimers in two states, representing steps either prior to or after passage through the DNA-gate. The structures locate the T-segment in important intermediate conformations of the catalytic cycle and provide insights into gyrase-DNA interactions and mechanism.

scholarly journals CryoEM structures of open dimers of gyrase A in complex with DNA illuminate mechanism of strand passage

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Katarzyna M Soczek ◽  
Tim Grant ◽  
Peter B Rosenthal ◽  
Alfonso Mondragón
Keyword(s):  
Conformational Changes ◽  
Dna Cleavage ◽  
Atp Hydrolysis ◽  
Unique Type ◽  
Dna Molecule ◽  
Gyrase A ◽  
A Subunit ◽  
Important Intermediate ◽  
Dna Strand ◽  
Strand Passage

Gyrase is a unique type IIA topoisomerase that uses ATP hydrolysis to maintain the negatively supercoiled state of bacterial DNA. In order to perform its function, gyrase undergoes a sequence of conformational changes that consist of concerted gate openings, DNA cleavage, and DNA strand passage events. Structures where the transported DNA molecule (T-segment) is trapped by the A subunit have not been observed. Here we present the cryoEM structures of two oligomeric complexes of open gyrase A dimers and DNA. The protein subunits in these complexes were solved to 4 Å and 5.2 Å resolution. One of the complexes traps a linear DNA molecule, a putative T-segment, which interacts with the open gyrase A dimers in two states, representing steps either prior to or after passage through the DNA-gate. The structures locate the T-segment in important intermediate conformations of the catalytic cycle and provide insights into gyrase-DNA interactions and mechanism.

scholarly journals Hindering the Strand Passage Reaction of Human Topoisomerase IIα without Disturbing DNA Cleavage, ATP Hydrolysis, or the Operation of the N-terminal Clamp

2004 ◽  
Vol 279 (27) ◽  
pp. 28093-28099 ◽  
Author(s):  
Vibe H. Oestergaard ◽  
Laura Giangiacomo ◽  
Lotte Bjergbaek ◽  
Birgitta R. Knudsen ◽  
Anni H. Andersen
Keyword(s):  
Dna Cleavage ◽  
Atp Hydrolysis ◽  
Topoisomerase Iiα ◽  
Strand Passage

Coupling ATP hydrolysis to DNA strand passage in type IIA DNA topoisomerases

2005 ◽  
Vol 33 (6) ◽  
pp. 1460 ◽  
Author(s):  
L. Costenaro ◽  
A. Maxwell ◽  
S. Mitelheiser ◽  
A.D. Bates
Keyword(s):  
Atp Hydrolysis ◽  
Dna Topoisomerases ◽  
Dna Strand ◽  
Strand Passage

scholarly journals Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F 1 -ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

2013 ◽  
Vol 12 (11) ◽  
pp. 1451-1461 ◽  
Author(s):  
Thuy La ◽  
George Desmond Clark-Walker ◽  
Xiaowen Wang ◽  
Stephan Wilkens ◽  
Xin Jie Chen
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Kluyveromyces Lactis ◽  
Β Subunit ◽  
Α Subunit ◽  
Content Type ◽  
Subunit Stoichiometry ◽  
Catalytic Sites ◽  
A Subunit

ABSTRACT F 1 -ATPase is a rotary molecular machine with a subunit stoichiometry of α 3 β 3 γ 1 δ 1 ε 1 . It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α 3 β 3 core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F 1 -ATPase have suggested that the α 3 β 3 core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F 1 -ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ o ) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F 1 complexes retained 21.7 to 44.6% of the native F 1 -ATPase activity. The γ-less F 1 subcomplex was assembled but was structurally and functionally labile in vitro . Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ o conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α 3 β 3 subcomplex.

Making the cut(s): how Cas12a cleaves target and non-target DNA

2019 ◽  
Vol 47 (5) ◽  
pp. 1499-1510 ◽  
Author(s):  
Daan C. Swarts
Keyword(s):  
Conformational Changes ◽  
Dna Sequences ◽  
Dna Cleavage ◽  
Transcriptional Control ◽  
Molecular Mechanisms ◽  
Nucleic Acid Detection ◽  
Target Dna ◽  
Activated State ◽  
Dna Strand ◽  
Cis And Trans

Abstract CRISPR–Cas12a (previously named Cpf1) is a prokaryotic deoxyribonuclease that can be programmed with an RNA guide to target complementary DNA sequences. Upon binding of the target DNA, Cas12a induces a nick in each of the target DNA strands, yielding a double-stranded DNA break. In addition to inducing cis-cleavage of the targeted DNA, target DNA binding induces trans-cleavage of non-target DNA. As such, Cas12a–RNA guide complexes can provide sequence-specific immunity against invading nucleic acids such as bacteriophages and plasmids. Akin to CRISPR–Cas9, Cas12a has been repurposed as a genetic tool for programmable genome editing and transcriptional control in both prokaryotic and eukaryotic cells. In addition, its trans-cleavage activity has been applied for high-sensitivity nucleic acid detection. Despite the demonstrated value of Cas12a for these applications, the exact molecular mechanisms of both cis- and trans-cleavage of DNA were not completely understood. Recent studies have revealed mechanistic details of Cas12a-mediates DNA cleavage: base pairing of the RNA guide and the target DNA induces major conformational changes in Cas12a. These conformational changes render Cas12a in a catalytically activated state in which it acts as deoxyribonuclease. This deoxyribonuclease activity mediates cis-cleavage of the displaced target DNA strand first, and the RNA guide-bound target DNA strand second. As Cas12a remains in the catalytically activated state after cis-cleavage, it subsequently demonstrates trans-cleavage of non-target DNA. Here, I review the mechanistic details of Cas12a-mediated cis- and trans-cleavage of DNA. In addition, I discuss how bacteriophage-derived anti-CRISPR proteins can inhibit Cas12a activity.

scholarly journals The role of ATP in the function of human ORC4 protein

2010 ◽  
Vol 75 (3) ◽  
pp. 317-322
Author(s):  
Aleksandra Divac ◽  
Branko Tomic ◽  
Jelena Kusic
Keyword(s):  
Conformational Changes ◽  
Origin Recognition Complex ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Protein A ◽  
Structural Role ◽  
A Subunit ◽  
Dna Substrates ◽  
Origin Recognition

Human ORC4 protein, a subunit of the origin recognition complex, belongs to the AAA+ superfamily of ATPases. Proteins belonging to this family require ATP for their function and interactions with ATP lead to conformational changes in them or in their partners. Human ORC4 protein induces structural changes in DNA substrates, promoting renaturation and formation of non-canonical structures, as well as conversion of single-stranded into multi-stranded oligonucleotide structures. The aim of this study was to further investigate the role of ATP in the function of human ORC4 protein. For this purpose, a mutant in the conserved Walker B motif of ORC4, which is able to bind but not to hydrolyze ATP, was constructed and its activity in DNA restructuring reactions was investigated. The obtained results showed that ATP hydrolysis is not necessary for the function of human ORC4. It is proposed that ATP has a structural role as a cofactor in the function of human ORC4 as a DNA restructuring agent.

scholarly journals Direct Observation of Topoisomerase IA Gate Dynamics

2018 ◽  
Author(s):  
Maria Mills ◽  
Yuk-Ching Tse-Dinh ◽  
Keir C. Neuman
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Mechanistic Model ◽  
Cellular Functions ◽  
Single Stranded Dna ◽  
Gate Opening ◽  
Type Ia ◽  
Dna Strand ◽  
Dynamics Simulations ◽  
Strand Passage

AbstractType IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although it is assumed type IA topoisomerases accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the E. coli type IA topoisomerases I and III. We found that following cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allow us to develop a mechanistic model of type IA topoisomerase-ssDNA interactions.

scholarly journals The pentapeptide-repeat protein, MfpA, interacts with mycobacterial DNA gyrase as a DNA T-segment mimic

2021 ◽  
Vol 118 (11) ◽  
pp. e2016705118
Author(s):  
Lipeng Feng ◽  
Julia E. A. Mundy ◽  
Clare E. M. Stevenson ◽  
Lesley A. Mitchenall ◽  
David M. Lawson ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Dna Cleavage ◽  
Atp Hydrolysis ◽  
Mutational Analysis ◽  
Dna Gyrase ◽  
Dna Breaks ◽  
Repeat Protein ◽  
X Ray Crystallography ◽  
Gyrase A ◽  
Negative Supercoiling

DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmatis gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA–MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins.

Synthesis and Characterisation of RuII Polypyridyl Complexes: DNA-Binding, Photocleavage, and Topoisomerase I and II Inhibitory Activity

2013 ◽  
Vol 66 (11) ◽  
pp. 1406 ◽  
Author(s):  
Xiaojun He ◽  
Guang Yang ◽  
Xiaonan Sun ◽  
Lingjun Xie ◽  
Lifeng Tan
Keyword(s):  
Dna Binding ◽  
Dna Cleavage ◽  
Topoisomerase I ◽  
Mixed Ligand ◽  
Dual Inhibitor ◽  
Cleavage Activity ◽  
Dna Cleavage Activity ◽  
Pbr322 Dna ◽  
Dna Strand ◽  
Strand Passage

Two mixed-ligand ruthenium(ii) complexes [Ru(phen)2(cptcp)]2+ (Ru1; phen = 1,10-phenanthroline, cptcp = 2-(4-carbazol-9-yl-phenyl)-1H-1,3,7,8-tetraaza-cyclopenta-[l]-phenanthrene) and [Ru(phen)2(btcpc)]2+ (Ru2; btcpc = 9-butyl-6-(1H-1,3,7,8-tetraaza-cyclo-cyclopenta-[l]-phenanthren-2-yl)-9H-carbazole-3-carbaldehyde) have been synthesised and characterised. The DNA-binding behaviours of the two complexes have been investigated by using spectroscopic and viscosity measurements. Results suggest that the two complexes bind to DNA by intercalation. The photocleavage of plasmid pBR322 DNA indicates that Ru1 exhibits more effective DNA cleavage activity in comparison to that exhibited by Ru2 under the same conditions, and different cleavage mechanisms are determined. Topoisomerase inhibition and DNA strand passage assay confirm that Ru1 may act as an efficient dual inhibitor of topoisomerases I and II, whereas Ru2 may only act as a single inhibitor of topoisomerases II.

scholarly journals Purification, crystallization and preliminary X-ray crystallographic studies of theMycobacterium tuberculosisDNA gyrase CTD

2012 ◽  
Vol 68 (2) ◽  
pp. 178-180 ◽  
Author(s):  
Amélie Darmon ◽  
Jérémie Piton ◽  
Mélanie Roué ◽  
Stéphanie Petrella ◽  
Alexandra Aubry ◽  
...  
Keyword(s):  
Unique Feature ◽  
Dna Gyrase ◽  
Type Ii ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unique Type ◽  
X Ray ◽  
His Tag ◽  
Gyrase A ◽  
A Subunit

Mycobacterium tuberculosisDNA gyrase, a nanomachine involved in regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolone in the treatment of tuberculosis. The C-terminal domain (CTD) of the DNA gyrase A subunit possesses a unique feature, the ability to wrap DNA in a chiral manner, that plays an essential role during the catalytic cycle. A construct of 36 kDa corresponding to this domain has been overproduced, purified and crystallized. Diffraction data were collected to 1.55 Å resolution. Cleavage of the N-terminal His tag was crucial for obtaining crystals. The crystals belonged to space groupP212121, with one molecule in the asymmetric unit and a low solvent content (33%). This is the first report of the crystallization and preliminary X-ray diffraction studies of a DNA gyrase CTD from a species that contains one unique type II topoisomerase.

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