scholarly journals Structure of telomerase-bound CST with Polymerase α-Primase

2021 ◽  
Author(s):  
Yao He ◽  
Song He ◽  
Henry Chan ◽  
Yaqiang Wang ◽  
Baocheng Liu ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Structural Basis ◽  
Binding Motif ◽  
Telomerase Reverse Transcriptase ◽  
Telomeric Dna ◽  
Double Stranded Dna ◽  
Key Players ◽  
Single Complex ◽  
Linear Chromosomes

Telomeres are the physical ends of linear chromosomes, composed of short repeating sequences (e.g. TTGGGG in Tetrahymena for the G-strand) of double-stranded DNA with a single-strand 3'-overhang of the G-strand and a group of proteins called shelterin. Among these, TPP1 and POT1 associate with the 3'-overhang, with POT1 binding the G-strand and TPP1 recruiting telomerase via interaction with telomerase reverse transcriptase (TERT). The ends of the telomeric DNA are replicated and maintained by telomerase, for the G-strand, and subsequently DNA Polymerase α-Primase (PolαPrim), for the C-strand. PolαPrim is stimulated by CTC1-STN1-TEN1 (CST), but the structural basis of both PolαPrim and CST recruitment to telomere ends remains unknown. Here we report cryo-EM structures of Tetrahymena CST in the context of telomerase holoenzyme, both in the absence and presence of PolαPrim, as well as of PolαPrim alone. Ctc1 binds telomerase subunit p50, a TPP1 ortholog, on a flexible Ctc1 binding motif unveiled jointly by cryo-EM and NMR spectroscopy. PolαPrim subunits are arranged in a catalytically competent conformation, in contrast to previously reported autoinhibited conformation. Polymerase POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. Together, we obtained a snapshot of four key players required for telomeric DNA synthesis in a single complex-telomerase core RNP, p50/TPP1, CST and PolαPrim-that provides unprecedented insights into CST and PolαPrim recruitment and handoff between G-strand and C-strand synthesis.

scholarly journals Structural insights into the promutagenic bypass of the major cisplatin-induced DNA lesion

2020 ◽  
Vol 477 (5) ◽  
pp. 937-951
Author(s):  
Hala Ouzon-Shubeita ◽  
Caroline K. Vilas ◽  
Seongmin Lee
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Cisplatin Resistance ◽  
Structural Basis ◽  
Abasic Site ◽  
Dna Polymerase Β ◽  
Dna Lesion ◽  
Human Dna ◽  
Optimal Conformation ◽  
Structural Insights

The cisplatin-1,2-d(GpG) (Pt-GG) intrastrand cross-link is the predominant DNA lesion generated by cisplatin. Cisplatin has been shown to predominantly induce G to T mutations and Pt-GG permits significant misincorporation of dATP by human DNA polymerase β (polβ). In agreement, polβ overexpression, which is frequently observed in cancer cells, is linked to cisplatin resistance and a mutator phenotype. However, the structural basis for the misincorporation of dATP opposite Pt-GG is unknown. Here, we report the first structures of a DNA polymerase inaccurately bypassing Pt-GG. We solved two structures of polβ misincorporating dATP opposite the 5′-dG of Pt-GG in the presence of Mg2+ or Mn2+. The Mg2+-bound structure exhibits a sub-optimal conformation for catalysis, while the Mn2+-bound structure is in a catalytically more favorable semi-closed conformation. In both structures, dATP does not form a coplanar base pairing with Pt-GG. In the polβ active site, the syn-dATP opposite Pt-GG appears to be stabilized by protein templating and pi stacking interactions, which resembles the polβ-mediated dATP incorporation opposite an abasic site. Overall, our results suggest that the templating Pt-GG in the polβ active site behaves like an abasic site, promoting the insertion of dATP in a non-instructional manner.

scholarly journals Structural basis for processive DNA synthesis by yeast DNA polymerase ε

2014 ◽  
Vol 70 (a1) ◽  
pp. C200-C200
Author(s):  
Matthew Hogg ◽  
Pia Osterman ◽  
Göran Bylund ◽  
Rais Ganai ◽  
Else-Britt Lundström ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Structural Basis ◽  
High Fidelity ◽  
Hairpin Loop ◽  
Catalytic Core ◽  
Double Stranded Dna ◽  
Eukaryotic Dna ◽  
Leading Strand ◽  
P Domain ◽  
Selection Of

DNA polymerase ε (Pol ε) is a high-fidelity polymerase that participates in leading-strand synthesis during eukaryotic DNA replication in eukaryotic cells. The 2.2 Å ternary structure of the 142 kDa catalytic core of Pol ε from Saccharomyces cerevisiae in complex with DNA and an incoming nucleotide has recently been determined [1]. The structure provides information about the selection of the correct nucleotide and the positions of amino acids that might be critical for proofreading activity. Pol ε has the highest fidelity among B-family polymerases despite the absence of an extended β-hairpin loop that is required for high-fidelity replication by other B-family polymerases. Moreover, the catalytic core has a new domain (i.e. the P-domain) that allows Pol ε to encircle the nascent double-stranded DNA and enhance processifivity of the polymerase. The structure provides valuable insights into the similarities and differences between Pol ε and other B-family polymerases, and suggests possible mechanisms responsible for the high processivity and fidelity of Pol ε.

scholarly journals Structural basis of DNA synthesis opposite 8-oxoguanine by human PrimPol primase-polymerase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Olga Rechkoblit ◽  
Robert E. Johnson ◽  
Yogesh K. Gupta ◽  
Louise Prakash ◽  
Satya Prakash ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Thermodynamic Stability ◽  
Active Site ◽  
Base Pair ◽  
Translesion Synthesis ◽  
Human Cells ◽  
Structural Basis ◽  
Human Dna ◽  
Dna Template

AbstractPrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3′−terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3′-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells.

scholarly journals Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid

2021 ◽  
Author(s):  
Tyler L Dangerfield ◽  
Serdal Kirmizialtin ◽  
Kenneth A. Johnson
Keyword(s):  
Amino Acid ◽  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerases ◽  
Conformational Dynamics ◽  
Unnatural Amino Acid ◽  
Structural Basis ◽  
High Fidelity ◽  
Active Site Residues ◽  
Kinetic Measurements

High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of different steps on specificity. During mismatch incorporation and subsequent extension (with the correct nucleotide), the rates of the conformational change and chemistry are both greatly reduced. The nucleotide dissociation rate, however, increases to greatly exceed the rate of chemistry. To investigate the structural basis for discrimination against mismatched nucleotides, we performed all atom molecular dynamics simulations on complexes with either the correct or mismatched nucleotide bound at the polymerase active site. We show that the closed form of the enzyme with a mismatch bound is greatly destabilized due to weaker interactions with active site residues, non-ideal base pairing, and a large increase in the distance from the 3′-OH group of the primer strand to the α-phosphate of the incoming nucleotide, explaining the reduced rates of misincorporation. The observed kinetic and structural mechanisms governing nucleotide misincorporation reveal the general principles likely applicable to other high fidelity DNA polymerases.

scholarly journals Crystal Structure of Butyrate Kinase 2 from Thermotoga maritima, a Member of the ASKHA Superfamily of Phosphotransferases

2009 ◽  
Vol 191 (8) ◽  
pp. 2521-2529 ◽  
Author(s):  
Jiasheng Diao ◽  
Miriam S. Hasson
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Active Site ◽  
Thermotoga Maritima ◽  
Structural Basis ◽  
Acetate Kinase ◽  
Phosphoryl Transfer ◽  
Atp Binding ◽  
Binding Motif ◽  
Butyrate Kinase

ABSTRACT The enzymatic transfer of phosphoryl groups is central to the control of many cellular processes. One of the phosphoryl transfer mechanisms, that of acetate kinase, is not completely understood. Besides better understanding of the mechanism of acetate kinase, knowledge of the structure of butyrate kinase 2 (Buk2) will aid in the interpretation of active-site structure and provide information on the structural basis of substrate specificity. The gene buk2 from Thermotoga maritima encodes a member of the ASKHA (acetate and sugar kinases/heat shock cognate/actin) superfamily of phosphotransferases. The encoded protein Buk2 catalyzes the phosphorylation of butyrate and isobutyrate. We have determined the 2.5-Å crystal structure of Buk2 complexed with (β,γ-methylene) adenosine 5′-triphosphate. Buk2 folds like an open-shelled clam, with each of the two domains representing one of the two shells. In the open active-site cleft between the N- and C-terminal domains, the active-site residues consist of two histidines, two arginines, and a cluster of hydrophobic residues. The ATP binding region of Buk2 in the C-terminal domain consists of abundant glycines for nucleotide binding, and the ATP binding motif is similar to those of other members of the ASKHA superfamily. The enzyme exists as an octamer, in which four disulfide bonds form between intermolecular cysteines. Sequence alignment and structure superposition identify the simplicity of the monomeric Buk2 structure, a probable substrate binding site, the key residues in catalyzing phosphoryl transfer, and the substrate specificity differences among Buk2, acetate, and propionate kinases. The possible enzyme mechanisms are discussed.

scholarly journals Cryo-EM structure of Pol κ−DNA−PCNA holoenzyme and implications for polymerase switching in DNA lesion bypass

2020 ◽  
Author(s):  
Claudia Lancey ◽  
Muhammad Tehseen ◽  
Masateru Takahashi ◽  
Mohamed A. Sobhy ◽  
Timothy J. Ragan ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Genome Instability ◽  
Structural Basis ◽  
Conformational Sampling ◽  
Lesion Bypass ◽  
Catalytic Core ◽  
Dna Lesion ◽  
Thumb Domain ◽  
C Terminus

Replacement of the stalled replicative polymerase (Pol δ) at a DNA lesion by the error-prone DNA polymerase κ (Pol κ) restarts synthesis past the lesion to prevent genome instability. The switching from Pol δ to Pol κ is mediated by the processivity clamp PCNA but the structural basis of this mechanism is unknown. We determined the Cryo-EM structures of human Pol κ–DNA–PCNA complex and of a stalled Pol δ–DNA–PCNA complex at 3.9 and 4.7 Å resolution, respectively. In Pol κ complex, the C-terminus of the PAD domain docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting Pol κ active site through PCNA. In Pol δ complex, the DNA is disengaged from the active site but is retained by the thumb domain. We present a model for polymerase switching facilitated by Pol κ recruitment to PCNA and Pol κ conformational sampling to seize the DNA from stalled Pol δ assisted by PCNA tilting.

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.

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