scholarly journals Mechanistic insights from structure of Mycobacterium smegmatis topoisomerase I with ssDNA bound to both N- and C-terminal domains

2020 ◽  
Vol 48 (8) ◽  
pp. 4448-4462 ◽  
Author(s):  
Nan Cao ◽  
Kemin Tan ◽  
Xiaobing Zuo ◽  
Thirunavukkarasu Annamalai ◽  
Yuk-Ching Tse-Dinh
Keyword(s):  
Mycobacterium Smegmatis ◽  
Topoisomerase I ◽  
Physiological Role ◽  
Opposite Polarity ◽  
Ssdna Binding ◽  
Binding Domains ◽  
Type Ia ◽  
Opposite Strand ◽  
Negative Supercoiling ◽  
Terminal Domains

Abstract Type IA topoisomerases interact with G-strand and T-strand ssDNA to regulate DNA topology. However, simultaneous binding of two ssDNA segments to a type IA topoisomerase has not been observed previously. We report here the crystal structure of a type IA topoisomerase with ssDNA segments bound in opposite polarity to the N- and C-terminal domains. Titration of small ssDNA oligonucleotides to Mycobacterium smegmatis topoisomerase I with progressive C-terminal deletions showed that the C-terminal region has higher affinity for ssDNA than the N-terminal active site. This allows the C-terminal domains to capture one strand of underwound negatively supercoiled DNA substrate first and position the N-terminal domains to bind and cleave the opposite strand in the relaxation reaction. Efficiency of negative supercoiling relaxation increases with the number of domains that bind ssDNA primarily with conserved aromatic residues and possibly with assistance from polar/basic residues. A comparison of bacterial topoisomerase I structures showed that a conserved transesterification unit (N-terminal toroid structure) for cutting and rejoining of a ssDNA strand can be combined with two different types of C-terminal ssDNA binding domains to form diverse bacterial topoisomerase I enzymes that are highly efficient in their physiological role of preventing excess negative supercoiling in the genome.

scholarly journals Stabilization of a G-Quadruplex from Unfolding by Replication Protein A Using Potassium and the Porphyrin TMPyP4

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Aishwarya Prakash ◽  
Fabien Kieken ◽  
Luis A. Marky ◽  
Gloria E. O. Borgstahl
Keyword(s):  
Dna Replication ◽  
Protein A ◽  
Thermal Stabilization ◽  
Replication Protein A ◽  
Replication Protein ◽  
Unique Problem ◽  
Ssdna Binding ◽  
Binding Domains ◽  
G Quadruplex ◽  
Stabilization Effect

Replication protein A (RPA) plays an essential role in DNA replication by binding and unfolding non-canonical single-stranded DNA (ssDNA) structures. Of the six RPA ssDNA binding domains (labeled A-F), RPA-CDE selectively binds a G-quadruplex forming sequence (5′-TAGGGGAAGGGTTGGAGTGGGTT-3′called Gq23). In K+, Gq23 forms a mixed parallel/antiparallel conformation, and in Na+Gq23 has a less stable (TMlowered by ∼20∘C), antiparallel conformation. Gq23 is intramolecular and 1D NMR confirms a stable G-quadruplex structure in K+. Full-length RPA and RPA-CDE-core can bind and unfold the Na+form of Gq23 very efficiently, but complete unfolding is not observed with the K+form. Studies with G-quadruplex ligands, indicate that TMPyP4 has a thermal stabilization effect on Gq23 in K+, and inhibits complete unfolding by RPA and RPA-CDE-core. Overall these data indicate that G-quadruplexes present a unique problem for RPA to unfold and ligands, such as TMPyP4, could possibly hinder DNA replication by blocking unfolding by RPA.

scholarly journals Deletion of the rpoZ gene, encoding the ω subunit of RNA polymerase, results in pleiotropic surface-related phenotypes in Mycobacterium smegmatis

Microbiology ◽  
2006 ◽  
Vol 152 (6) ◽  
pp. 1741-1750 ◽  
Author(s):  
Renjith Mathew ◽  
Raju Mukherjee ◽  
Radhakrishnan Balachandar ◽  
Dipankar Chatterji
Keyword(s):  
Rna Polymerase ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Physiological Role ◽  
Wild Type ◽  
Gene Encoding ◽  
Biofilm Growth ◽  
Mutant Bacterium ◽  
Sliding Motility

The ω subunit, the smallest subunit of bacterial RNA polymerase, is known to be involved in maintaining the conformation of the β′ subunit and aiding its recruitment to the rest of the core enzyme assembly in Escherichia coli. It has recently been shown in Mycobacterium smegmatis, by creating a deletion mutation of the rpoZ gene encoding ω, that the physiological role of the ω subunit also includes providing physical protection to β′. Interestingly, the mutant had altered colony morphology. This paper demonstrates that the mutant mycobacterium has pleiotropic phenotypes including reduced sliding motility and defective biofilm formation. Analysis of the spatial arrangement of biofilms by electron microscopy suggests that the altered phenotype of the mutant arises from a deficiency in generation of extracellular matrix. Complementation of the mutant strain with a copy of the wild-type rpoZ gene integrated in the bacterial chromosome restored both sliding motility and biofilm formation to the wild-type state, unequivocally proving the role of ω in the characteristics observed for the mutant bacterium. Analysis of the cell wall composition demonstrated that the mutant bacterium had an identical glycopeptidolipid profile to the wild-type, but failed to synthesize the short-chain mycolic acids characteristic of biofilm growth in M. smegmatis.

scholarly journals Functional domain organization of the potato α-glucan, water dikinase (GWD): evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants

2005 ◽  
Vol 385 (2) ◽  
pp. 355-361 ◽  
Author(s):  
René MIKKELSEN ◽  
Andreas BLENNOW
Keyword(s):  
Structural Changes ◽  
Sequence Similarity ◽  
Limited Proteolysis ◽  
Phosphoryl Group ◽  
Deletion Mutants ◽  
Functional Domain ◽  
Partial Reaction ◽  
Atp Binding Site ◽  
Binding Domains ◽  
Terminal Domains

The potato tuber (Solanum tuberosum) GWD (α-glucan, water dikinase) catalyses the phosphorylation of starch by a dikinase-type reaction mechanism in which the β-phosphate of ATP is transferred to the glucosyl residue of amylopectin. GWD shows sequence similarity to bacterial pyruvate, water dikinase and PPDK (pyruvate, phosphate dikinase). In the present study, we examine the structure–function relationship of GWD. Analysis of proteolytic fragments of GWD, in conjunction with peptide microsequencing and the generation of deletion mutants, indicates that GWD is comprised of five discrete domains of 37, 24, 21, 36 and 38 kDa. The catalytic histidine, which mediates the phosphoryl group transfer from ATP to starch, is located on the 36 kDa fragment, whereas the 38 kDa C-terminal fragment contains the ATP-binding site. Binding of the glucan molecule appears to be confined to regions containing the three N-terminal domains. Deletion mutants were generated to investigate the functional interdependency of the putative ATP- and glucan-binding domains. A truncated form of GWD expressing the 36 and 38 kDa C-terminal domains was found to catalyse the E+ATP→E-P+AMP+Pi (where Pi stands for orthophosphate) partial reaction, but not the E-P+glucan→E+glucan-P partial reaction. CD experiments provided evidence for large structural changes on autophosphorylation of GWD, indicating that GWD employs a swivelling-domain mechanism for enzymic phosphotransfer similar to that seen for PPDK.

Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I

2009 ◽  
Vol 393 (4) ◽  
pp. 788-802 ◽  
Author(s):  
Anuradha Gopal Bhat ◽  
Majety Naga Leelaram ◽  
Shivanand Manjunath Hegde ◽  
Valakunja Nagaraja
Keyword(s):  
Catalytic Activity ◽  
Mycobacterium Smegmatis ◽  
Topoisomerase I ◽  
Metal Coordination ◽  
Distinct Role

scholarly journals Role of Histone N-Terminal Tails and Their Acetylation in Nucleosome Dynamics

2000 ◽  
Vol 20 (19) ◽  
pp. 7230-7237 ◽  
Author(s):  
Violette Morales ◽  
Hélène Richard-Foy
Keyword(s):  
Topoisomerase I ◽  
Globular Domain ◽  
Histone Tail ◽  
Histone Tails ◽  
Nucleosome Dynamics ◽  
Nucleosome Structure ◽  
Left Handed ◽  
Negative Supercoiling ◽  
And Function

ABSTRACT Histone N-terminal tails are central to the processes that modulate nucleosome structure and function. We have studied the contribution of core histone tails to the structure of a single nucleosome and to a histone (H3-H4)2 tetrameric particle assembled on a topologically constrained DNA minicircle. The effect of histone tail cleavage and histone tail acetylation on the structure of the nucleoprotein particle was investigated by analyzing the DNA topoisomer equilibrium after relaxation of DNA torsional stress by topoisomerase I. Removal of the H3 and H4 N-terminal tails, as well as their acetylation, provoked a dramatic change in the linking-number difference of the (H3-H4)2 tetrameric particle, with a release of up to 70% of the negative supercoiling previously constrained by this structure. The (H3-H4)2 tetramers containing tailless or hyperacetylated histones showed a striking preference for relaxed DNA over negatively supercoiled DNA. This argues in favor of a change in tetramer structure that constrains less DNA and adopts a relaxed flat conformation instead of its left-handed conformation within the nucleosome. In contrast neither removal or hyperacetylation of H3 and H4 tails nor removal or hyperacetylation of H2A and H2B N-terminal tails affected the nucleosome structure. This indicates that the globular domain of H2A and H2B is sufficient to stabilize the tailless or the hyperacetylated (H3-H4)2tetramer in a left-handed superhelix conformation. These results suggest that the effect of histone tail acetylation that facilitates transcription may be mediated via transient formation of an (H3-H4)2 tetrameric particle that could adopt an open structure only when H3 and/or H4 tails are hyperacetylated.

scholarly journals The Sbi Protein Is a Multifunctional Immune Evasion Factor of Staphylococcus aureus

2011 ◽  
Vol 79 (9) ◽  
pp. 3801-3809 ◽  
Author(s):  
Emma Jane Smith ◽  
Livia Visai ◽  
Steven W. Kerrigan ◽  
Pietro Speziale ◽  
Timothy J. Foster
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Evasion ◽  
Capsular Polysaccharide ◽  
Cell Envelope ◽  
Protein A ◽  
Biologically Active ◽  
Complement Protein ◽  
Content Type ◽  
Binding Domains ◽  
Terminal Domains

ABSTRACTThe second immunoglobulin-binding protein (Sbi) ofStaphylococcus aureushas two N-terminal domains that bind the Fc region of IgG in a fashion similar to that of protein A and two domains that can bind to the complement protein C3 and promote its futile consumption in the fluid phase. It has been proposed that Sbi helps bacteria to avoid innate immune defenses. By comparing a mutant defective in Sbi with mutants defective in protein A, clumping factor A, iron-regulated surface determinant H, and capsular polysaccharide, it was shown that Sbi is indeed an immune evasion factor that promotes bacterial survival in whole human blood and the avoidance of neutrophil-mediated opsonophagocytosis. Sbi is present in the culture supernatant and is also associated with the cell envelope.S. aureusstrains that expressed truncates of Sbi lacking N-terminal domains D1 and D2 (D1D2) or D3 and D4 (D3D4) or a C-terminal truncate that was no longer retained in the cell envelope were analyzed. Both the secreted and envelope-associated forms of Sbi contributed to immune evasion. The IgG-binding domains contributed only when Sbi was attached to the cell, while only the secreted C3-binding domains were biologically active.

scholarly journals Cloning, Tissue Expression, and Chromosomal Localization of the Mouse IRS-3 Gene

Endocrinology ◽  
1997 ◽  
Vol 138 (11) ◽  
pp. 4931-4940 ◽  
Author(s):  
Salvatore Sciacchitano ◽  
Simeon I. Taylor
Keyword(s):  
Intracellular Signaling ◽  
Messenger Rna ◽  
Expressed Sequence Tag ◽  
Physiological Role ◽  
Signaling Molecules ◽  
Cellular Growth ◽  
Telomeric Region ◽  
Pleckstrin Homology ◽  
Binding Domains ◽  
Expressed Sequence

Insulin receptor substrate (IRS) proteins are key regulators of basic functions such as cellular growth and metabolism. They provide an interface between multiple receptors and a complex network of intracellular signaling molecules. Two members of this family (IRS-1 and IRS-2) have been identified previously. In this investigation, we analyzed a mouse expressed sequence tag clone that proved to be a new member of the IRS family. Sequence analysis of this clone and comparison with the sequences deposited in GenBank demonstrates this protein may be the murine homolog of rat IRS-3, recently purified and cloned from rat adipocytes. Accordingly, we have named our protein mouse IRS-3. The expressed sequence tag clone contains the complete coding sequence of 1485 bp, encoding a protein of 495 amino acids. Sequence alignment with the other members of the IRS family shows that this protein contains pleckstrin homology and phosphotyrosine-binding domains that are highly conserved. In addition, there is conservation of many tyrosine phosphorylation motifs responsible for interactions with downstream signaling molecules containing SH2 domains. The murine IRS-3 messenger RNA (2.4 kilobases in length) is expressed in many tissues, with highest levels in liver and lung. Mouse IRS-3 is highly expressed in the first part of the embryonic life, when IRS-1 messenger RNA is barely detectable. Unlike the genes encoding IRS-1 and IRS-2, the IRS-3 gene contains an intron (344 bp in length) in the region between the pleckstrin homology and the phosphotyrosine-binding domains. Fluorescent in situ hybridization localized the mouse IRS-3 gene on the telomeric region of chromosome 5G2. Cloning of the murine IRS-3 gene will make it possible to apply genetic approaches to elucidate the physiological role of this new member of the IRS family of proteins.

scholarly journals Dynamics and Selective Remodeling of the DNA Binding Domains of RPA

2018 ◽  
Author(s):  
Nilisha Pokhrel ◽  
Colleen C. Caldwell ◽  
Elliot I. Corless ◽  
Emma A. Tillison ◽  
Joseph Tibbs ◽  
...  
Keyword(s):  
Dna Binding ◽  
Conformational Dynamics ◽  
Protein A ◽  
Interacting Proteins ◽  
Ssdna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Mediator Protein ◽  
Damage Response ◽  
The Individual

AbstractReplication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-strand DNA (ssDNA) intermediates, activating the DNA damage response, and handing off ssDNA to appropriate downstream players. Six DNA binding domains (DBDs) in RPA promote high affinity binding to ssDNA, but also allow RPA displacement by lower affinity proteins. We have made fluorescent versions of RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA, while RPA as a whole remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. This demonstrates how RPA interacting proteins, with lower ssDNA binding affinity, can access the occluded ssDNA and remodel individual DBDs to replace RPA.One Sentence SummaryThe choreography of binding and rearrangement of the individual domains of RPA during homologous recombination is revealed.

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