scholarly journals Structural intermediates of a DNA–ligase complex illuminate the role of the catalytic metal ion and mechanism of phosphodiester bond formation

2019 ◽  
Vol 47 (14) ◽  
pp. 7147-7162 ◽  
Author(s):  
Adele Williamson ◽  
Hanna-Kirsti S Leiros
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Bond Formation ◽  
Strand Break ◽  
Nucleophilic Attack ◽  
Dna Ligase ◽  
Phosphodiester Bond ◽  
Dna Ligases ◽  
Catalytic Metal ◽  
Ternary Structure

Abstract DNA ligases join adjacent 5′ phosphate (5′P) and 3′ hydroxyl (3′OH) termini of double-stranded DNA via a three-step mechanism requiring a nucleotide cofactor and divalent metal ion. Although considerable structural detail is available for the first two steps, less is known about step 3 where the DNA-backbone is joined or about the cation role at this step. We have captured high-resolution structures of an adenosine triphosphate (ATP)-dependent DNA ligase from Prochlorococcus marinus including a Mn-bound pre-ternary ligase–DNA complex poised for phosphodiester bond formation, and a post-ternary intermediate retaining product DNA and partially occupied AMP in the active site. The pre-ternary structure unambiguously identifies the binding site of the catalytic metal ion and confirms both its role in activating the 3′OH terminus for nucleophilic attack on the 5′P group and stabilizing the pentavalent transition state. The post-ternary structure indicates that DNA distortion and most enzyme-AMP contacts remain after phosphodiester bond formation, implying loss of covalent linkage to the DNA drives release of AMP, rather than active site rearrangement. Additionally, comparisons of this cyanobacterial DNA ligase with homologs from bacteria and bacteriophage pose interesting questions about the structural origin of double-strand break joining activity and the evolution of these ATP-dependent DNA ligase enzymes.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

scholarly journals Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured Using the α-Hemolysin Nanopore

ACS Nano ◽  
2016 ◽  
Vol 10 (12) ◽  
pp. 11127-11135 ◽  
Author(s):  
Cherie S. Tan ◽  
Jan Riedl ◽  
Aaron M. Fleming ◽  
Cynthia J. Burrows ◽  
Henry S. White
Keyword(s):  
Bond Formation ◽  
Dna Ligase ◽  
Phosphodiester Bond ◽  
Kinetics Of

scholarly journals Active-Site Models of Streptococcus pyogenes Cas9 in DNA Cleavage State

2021 ◽  
Vol 8 ◽  
Author(s):  
Honghai Tang ◽  
Hui Yuan ◽  
Wenhao Du ◽  
Gan Li ◽  
Dongmei Xue ◽  
...  
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Active Sites ◽  
Structural Similarity ◽  
Atomic Model ◽  
Nucleophilic Attack ◽  
Target Dna ◽  
Coordinated Water ◽  
Dna Complex

CRISPR-Cas9 is a powerful tool for target genome editing in living cells. Significant advances have been made to understand how this system cleaves target DNA. HNH is a nuclease domain, which shares structural similarity with the HNH endonuclease characterzied by a beta-beta-alpha-metal fold. Therefore, based on one- and two-metal-ion mechanisms, homology modeling and molecular dynamics (MD) simulation are suitable tools for building an atomic model of Cas9 in the DNA cleavage state. Here, by modeling and MD, we presented an atomic model of SpCas9–sgRNA–DNA complex with the cleavage state. This model shows that the HNH and RuvC conformations resemble their DNA cleavage state where the active-sites in the complex coordinate with DNA, Mg2+ ions, and water. Among them, residues D10, E762, H983, and D986 locate at the first shell of the RuvC active-site and interact with the ions directly, residues H982 or/and H985 are general (Lewis) bases, and the coordinated water is located at the positions for nucleophilic attack of the scissile phosphate. Meanwhile, this catalytic model led us to engineer a new SpCas9 variant (SpCas9-H982A + H983D) with reduced off-target effects. Thus, our study provided new mechanistic insights into the CRISPR-Cas9 system in the DNA cleavage state and offered useful guidance for engineering new CRISPR-Cas9 editing systems with improved specificity.

scholarly journals NAD+ is not utilized as a co-factor for DNA ligation by human DNA ligase IV

2020 ◽  
Vol 48 (22) ◽  
pp. 12746-12750
Author(s):  
Bailin Zhao ◽  
Tasmin Naila ◽  
Michael R Lieber ◽  
Alan E Tomkinson
Keyword(s):  
Strand Break ◽  
Dna Ligase ◽  
Unexpected Finding ◽  
Dna Ligases ◽  
Dna Ligase Iv ◽  
Human Dna ◽  
Dna Double Strand Break ◽  
Ligase Iv ◽  
Eukaryotic Dna ◽  
Catalytic Cycles

Abstract As nucleotidyl transferases, formation of a covalent enzyme-adenylate intermediate is a common first step of all DNA ligases. While it has been shown that eukaryotic DNA ligases utilize ATP as the adenylation donor, it was recently reported that human DNA ligase IV can also utilize NAD+ and, to a lesser extent ADP-ribose, as the source of the adenylate group and that NAD+, unlike ATP, enhances ligation by supporting multiple catalytic cycles. Since this unexpected finding has significant implications for our understanding of the mechanisms and regulation of DNA double strand break repair, we attempted to confirm that NAD+ and ADP-ribose can be used as co-factors by human DNA ligase IV. Here, we provide evidence that NAD+ does not enhance ligation by pre-adenylated DNA ligase IV, indicating that this co-factor is not utilized for re-adenylation and subsequent cycles of ligation. Moreover, we find that ligation by de-adenylated DNA ligase IV is dependent upon ATP not NAD+ or ADP-ribose. Thus, we conclude that human DNA ligase IV cannot use either NAD+ or ADP-ribose as adenylation donor for ligation.

Identification of an Active Site Ligand for a Group I Ribozyme Catalytic Metal Ion†

Biochemistry ◽  
2002 ◽  
Vol 41 (8) ◽  
pp. 2516-2525 ◽  
Author(s):  
Alexander A. Szewczak ◽  
Anne B. Kosek ◽  
Joseph A. Piccirilli ◽  
Scott A. Strobel
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Group I ◽  
Catalytic Metal

scholarly journals Thermodynamic Evidence for Negative Charge Stabilization by a Catalytic Metal Ion within an RNA Active Site

2011 ◽  
Vol 7 (2) ◽  
pp. 294-299 ◽  
Author(s):  
Raghuvir N. Sengupta ◽  
Daniel Herschlag ◽  
Joseph A. Piccirilli
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Negative Charge ◽  
Catalytic Metal ◽  
Charge Stabilization

scholarly journals Structure of human apurinic/apyrimidinic endonuclease 1 with the essential Mg2+cofactor

2013 ◽  
Vol 69 (12) ◽  
pp. 2555-2562 ◽  
Author(s):  
Brittney A. Manvilla ◽  
Edwin Pozharski ◽  
Eric A. Toth ◽  
Alexander C. Drohat
Keyword(s):  
Active Site ◽  
Excision Repair ◽  
Strand Break ◽  
Dnase I ◽  
Dna Lesions ◽  
Water Molecules ◽  
Repair Synthesis ◽  
Phosphodiester Bond ◽  
Abasic Sites ◽  
Catalytic Function

Apurinic/apyrimidinic endonuclease 1 (APE1) mediates the repair of abasic sites and other DNA lesions and is essential for base-excision repair and strand-break repair pathways. APE1 hydrolyzes the phosphodiester bond at abasic sites, producing 5′-deoxyribose phosphate and the 3′-OH primer needed for repair synthesis. It also has additional repair activities, including the removal of 3′-blocking groups. APE1 is a powerful enzyme that absolutely requires Mg2+, but the stoichiometry and catalytic function of the divalent cation remain unresolved for APE1 and for other enzymes in the DNase I superfamily. Previously reported structures of DNA-free APE1 contained either Sm3+or Pb2+in the active site. However, these are poor surrogates for Mg2+because Sm3+is not a cofactor and Pb2+inhibits APE1, and their coordination geometry is expected to differ from that of Mg2+. A crystal structure of human APE1 was solved at 1.92 Å resolution with a single Mg2+ion in the active site. The structure reveals ideal octahedral coordination of Mg2+viatwo carboxylate groups and four water molecules. One residue that coordinates Mg2+directly and two that bind inner-sphere water molecules are strictly conserved in the DNase I superfamily. This structure, together with a recent structure of the enzyme–product complex, inform on the stoichiometry and the role of Mg2+in APE1-catalyzed reactions.

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