Figure 6 - uploaded by Francisco J Esteban
Content may be subject to copyright.
+1 addition on a blunt-ended DNA substrate. The template-independent addition of a nucleotide on the blunt-ended sp1/sp1c (15/15mer) substrate was performed as described in Materials and Methods using Mg2+ as metal activator. The positions of the non-elongated primer (15mer), elongated primer (16mer) and degraded primer (3mer) can be observed on this autoradiograph of an 8 M urea–20% polyacrylamide gel.
Source publication
Alignment of the protein sequence of DNA-dependent DNA polymerases has allowed the definition of a new motif, lying adjacent
to motif B in the direction of the N‐terminus and therefore named pre-motif B. Both motifs are located in the fingers subdomain,
shown to rotate towards the active site to form a dNTP-binding pocket in several DNA polymerases...
Contexts in source publication
Context 1
... both mutant DNA polymerases, I364Q and K371T, were affected in non- templated formation of TP-dAMP, the similar reaction under DNA-primed conditions was studied. As can be seen in Figure 6, the wild-type DNA polymerase is able to perform +1 addition at high dNTP concentrations (Table 1). On the other hand, polymerisation from position 15 to 16 could not be observed with either of the two mutant DNA polymerases. ...
Context 2
... 6, the wild-type DNA polymerase is able to perform +1 addition at high dNTP concentrations (Table 1). On the other hand, polymerisation from position 15 to 16 could not be observed with either of the two mutant DNA polymerases. In comparison to the wild-type DNA polymerase the exonuclease activity of the mutant DNA polymerases on this substrate (Fig. 6, lane 0 µM dNTP) was similar to that on the template/ primer DNA ( Table 1), suggesting that their DNA-binding capacity was also similar for both substrates. When Mn 2+ was used as metal activator the wild-type DNA polymerase required a lower dNTP concentration to perform +1 addition (Table 1). Under these conditions mutant DNA ...
Similar publications
Human exonuclease 1 (EXO1), a 5′→3′ exonuclease, contributes to the regulation of the cell cycle checkpoints, replication fork maintenance, and post replicative DNA repair pathways. These processes are required for the resolution of stalled or blocked DNA replication that can lead to replication stress and potential collapse of the replication fork...
Duplication of bacterial chromosomes is initiated via the assembly of two replication forks at a single defined origin. Forks proceed bi-directionally until they fuse in a specialised termination area opposite the origin. This area is flanked by polar replication fork pause sites that allow forks to enter but not to leave. The precise function of t...
Replicative DNA polymerases (DNAPs) require divalent metal cations for phosphodiester bond formation in the polymerase site
and for hydrolytic editing in the exonuclease site. Me2+ ions are intimate architectural components of each active site, where
they are coordinated by a conserved set of amino acids and functional groups of the reaction substr...
Abasic sites represent the most frequent DNA lesions in the genome that have high mutagenic potential and lead to mutations commonly found in human cancers. Although these lesions are devoid of the genetic information, adenine is most efficiently inserted when abasic sites are bypassed by DNA polymerases, a phenomenon termed A-rule. In this study,...
Bacteriophage ϕ29 DNA polymerase has two activities: DNA polymerization and 3′-5′ exonucleolysis governed by catalytic sites present in two structurally distant domains. These domains must work together to allow the correct replication of the template and to prevent the accumulation of errors in the newly synthesized DNA strand. ϕ29 DNA polymerase...
Citations
... The library pre-selection (R0) was prepared and sent for deep sequencing as described in Section 2.4.1. [105,18]. This suggests that modifying K371 of phi29 DNAP does not improve recognition and incorporation of sugar-modified nucleosides but remains a potential candidate for the backbone-modified substrates such as phosphorothioate [21] and boranophosphate [22] nucleotides. ...
... The stringent selection (R1c4) enriched the wild-type isoleucine in position 6, corresponding to I364, a structurally relevant residue involved in DNA and dNTP binding [105], as well as I364Q, which has shown to reduce the exonuclease activity of phi29 DNAP [105]. In the longer and less stringent such as the nonlinear least-squares method [98] or other non-parametric tests such as the Kruskal-Wallis test [99] by ranks should be implemented to corroborate the previously mentioned observations. ...
... The stringent selection (R1c4) enriched the wild-type isoleucine in position 6, corresponding to I364, a structurally relevant residue involved in DNA and dNTP binding [105], as well as I364Q, which has shown to reduce the exonuclease activity of phi29 DNAP [105]. In the longer and less stringent such as the nonlinear least-squares method [98] or other non-parametric tests such as the Kruskal-Wallis test [99] by ranks should be implemented to corroborate the previously mentioned observations. ...
Xenobiotic nucleic acids are unnatural analogues of DNA (and RNA) of great biotechnological and pharmaceutical interest. Their chemical synthesis is highly challenging and expensive, rendering the discovery of functional XNAs impractical. Enzymatic synthesis of XNA is a viable alternative but is limited by the low efficiency and low substrate selectivity of the currently available engineered XNA polymerases. A better understanding of the functional space of polymerases is needed to improve their engineering. Directed evolution techniques were implemented to map the sequence-function relationships of phi29 DNA polymerase (phi29 DNAP) in the process of evolving a more efficient HNA polymerase. Phi29 DNAP is a small and well-characterised polymerase with remarkable processivity and limited HNA synthesis activity. Diversity in phi29 DNAP was introduced through insertions and deletions, multiple-site saturation mutagenesis and random mutagenesis, targeting different subdomains of the enzyme. Libraries were partitioned through compartmentalised self-tagging (CST), a functional selection platform for XNA synthetases. A single round of selection did not substantially alter the overall activity of libraries for HNA synthesis, and variants isolated in screening were of comparable activity to the wild-type enzyme. Nevertheless, the results give insight into the functional landscape of phi29 DNAP. Deep sequencing of the libraries used in selection was used to quantify the functional consequence of sequence variation and investigate signs of epistasis for HNA synthesis. Variants that were enriched more than the wild-type, with potentially enhanced HNA synthesis activity, as well as variants that were significantly depleted were identified, both of which contribute to our understanding of the functional sequence space of phi29 DNAP. Altogether, the directed evolution and deep mutational scanning approaches implemented could be used to develop a new generation of more efficient XNA polymerases.
... As mention before, we initially excluded the possibility that translocation may occur associated to the rate limiting step. This assumption was made based on extensive biochemical and structural data showing that no significant conformational changes within the polymerase-DNA complex occur during this reaction (6,(14)(15)(16)(17). Interestingly, as shown before, the results from the fits directly corroborated that the rate limiting step of the nucleotide incorporation cycle does not depend on force and therefore, it is not associated with translocation. ...
... Finally, to study the process of product release we measured the effect of PPi on replication velocity at a subsaturating dNTP concentration of 2 M. Increasing the PPi concentration 1000-fold (to 1 mM) had no significant effect on the average replication velocity measured at different loads (Supplementary Figure S3). In agreement with bulk studies, these results indicate that under our experimental conditions the equilibrium constant for PPi release is very large (52,53). Accordingly, this step was considered largely irreversible for all the mechano-chemical models contemplated in this work ( Figure 1B-D). ...
... To determine the load-dependent step, or the step of the nucleotide incorporation reaction related to movement, we considered a minimal nucleotide incorporation cycle where the rate-limiting activation of the ternary complex and the following rapid chemical steps were grouped within a single rate limiting step (k cat , Figure 1A). According to biochemical and structural studies no significant conformational changes within the polymerase-DNA complex occur during these steps (2,4,21,24,52,54), indicating that translocation is not expected to occur concomitantly to these reactions. Hence, in this minimal nucleotide incorporation cycle ( Figure 1A), three different general models might explain the coupling mechanism between the chemical and mechanical steps of the reaction. ...
During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN).
© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
... In the case of 29, up to now only mutants in DNA polymerase residues had been shown to affect the kinetics of initiation (51)(52)(53)(54)(55). The results presented in this paper show the involvement of 29 TP N-terminal domain DNA binding residues in the kinetics of initiation of viral DNA replication and stresses the importance of the TP N-terminal domain both for a proper interaction with the DNA polymerase and for TP-DNA amplification in vitro. ...
Phage ϕ29 DNA replication takes place by a protein-priming mechanism in which the viral DNA polymerase catalyses the covalent linkage of the initiating nucleotide to a specific serine residue of the terminal protein (TP). The N-terminal domain of the ϕ29 TP has been shown to bind to the host DNA in a sequence-independent manner and this binding is essential for the TP nucleoid localisation and for an efficient viral DNA replication in vivo. In the present work we have studied the involvement of the TP N-terminal domain residues responsible for DNA binding in the different stages of viral DNA replication by assaying the in vitro activity of purified TP N-terminal mutant proteins. The results show that mutation of TP residues involved in DNA binding affects the catalytic activity of the DNA polymerase in initiation, as the Km for the initiating nucleotide is increased when these mutant proteins are used as primers. Importantly, this initiation defect was relieved by using the ϕ29 double-stranded DNA binding protein p6 in the reaction, which decreased the Km of the DNA polymerase for dATP about 130-190 fold. Furthermore, the TP N-terminal domain was shown to be required both for a proper interaction with the DNA polymerase and for an efficient viral DNA amplification.
© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
... Crystallographic data are from PDB1 IG9 (RB69 DNA polymerase) and PDB1 3KTQ (Taq DNA polymerase). a nearly invariant Lys in family B DNA polymerases 8,23 (His639 of Taq and Lys486 of RB69 DNA polymerases, see Figure 1). Mutational analysis of these two Lys residues of RB69 DNA polymerase located in motif B and pre-motif B has supported their importance in dNTP binding. ...
... In agreement with this, site-directed mutagenesis performed with f29 DNA polymerase, belonging to the family B DNA polymerases able to start replication by protein-priming, has supported experimentally the roles in dNTP binding of the Lys of motif B (Lys383) 18 and the Lys of pre-motif B (Lys371). 23 Since in the protein sequences of the DNA polymerases of this subgroup no positively charged amino acid residue can be found at the position corresponding to the Arg of pre-motif B, but positively charged amino acid residues can be found at the position corresponding to the Arg of motif B, we have addressed here the question if f29 DNA polymerase has more amino acid residues involved in nucleotide binding besides Lys371 and Lys383. We can conclude that residue Lys379 of motif B, conserved in protein-primed DNA polymerases and located at the position corresponding to the invariant Arg involved in dNTP binding in family A DNA polymerases, is also involved in binding the incoming nucleotide in f29 DNA polymerase. ...
... Previously studied residues of f29 DNA polymerase in this region are indicated with an asterisk. 17,18,23,59 The residues studied here, Lys366 and Lys379, and the other residues known to be involved in nucleotide binding through the phosphate group 23 are additionally indicated with black and grey arrows, respectively. The following conserved amino acid residues were considered: S and T; A and G; D and E; K, R and H; A, I, L, M, C and V; Y and F. concentrations are required for the wild-type f29 DNA polymerase to: (1) start replication and polymerise the first nucleotide (10 nM, from position 15 to 16); (2) to compete effectively the 3 0 -5 0 -exonuclease activity replicating until the penultimate nucleotide (30 nM, from position 16 to 20); (3) to replicate the last nucleotide of the primer/template substrate (10 mM, from position 20 to 21). ...
... 16 properties adopted from Syed et al.[20]and one additional property) were calculated. The importance of negatively/positively charged residues in protein function has been described in several studies[20,23,24,38,60]. The 20 standard amino acids are divided into negatively charged residues, positively charged residues, and neutral residues according to their pI. ...
Predicting the function of an unknown protein is an essential goal in bioinformatics. Sequence similarity-based approaches are widely used for function prediction; however, they are often inadequate in the absence of similar sequences or when the sequence similarity among known protein sequences is statistically weak. This study aimed to develop an accurate prediction method for identifying protein function, irrespective of sequence and structural similarities.
A highly accurate prediction method capable of identifying protein function, based solely on protein sequence properties, is described. This method analyses and identifies specific features of the protein sequence that are highly correlated with certain protein functions and determines the combination of protein sequence features that best characterises protein function. Thirty-three features that represent subtle differences in local regions and full regions of the protein sequences were introduced. On the basis of 484 features extracted solely from the protein sequence, models were built to predict the functions of 11 different proteins from a broad range of cellular components, molecular functions, and biological processes. The accuracy of protein function prediction using random forests with feature selection ranged from 94.23% to 100%. The local sequence information was found to have a broad range of applicability in predicting protein function.
We present an accurate prediction method using a machine-learning approach based solely on protein sequence properties. The primary contribution of this paper is to propose new PNPRD features representing global and/or local differences in sequences, based on positively and/or negatively charged residues, to assist in predicting protein function. In addition, we identified a compact and useful feature subset for predicting the function of various proteins. Our results indicate that sequence-based classifiers can provide good results among a broad range of proteins, that the proposed features are useful in predicting several functions, and that the combination of our and traditional features may support the creation of a discriminative feature set for specific protein functions.
... The phosphates of the incoming dNTP interact with the basic side chains of residues K371 and K383 from the fingers subdomain. Consistent with mutational data, the conserved sequence motif B residue K383 (Saturno et al, 1997) interacts with the a-and g-phosphates of the incoming dNTP, and the pre-B motif residue K371 (Truniger et al, 2002) interacts with the g-phosphate (Figure 2B). It is possible that these residues comprise part of a pre-insertion-binding site for the nucleotide , as, in the apo structure, they were observed to interact with sulfate ions which are sterically and electrostatically similar to the phosphate groups of a nucleotide (PDBID: 1XHX) (Kamtekar et al, 2004), although kinetics experiments with the B-family DNAP from bacteriophage RB69 have been interpreted to indicate the absence of a pre-insertion site in that system (Yang et al, 2002b). ...
... From these studies, Yin and Steitz (2004) predicted a similar mechanism for all polymerases that undergo a conformational change in response to nucleotide binding. Assuming, as mutational data indicate, that the chemical step does not drastically alter the conformations of amino acids at the active site of B-family polymerases (Truniger et al, 2002 ), our structures of the binary and ternary complexes of phi29 DNAP provide a basis for proposing a structural mechanism of translocation for the B-family of polymerases. In this mechanism, in phi29 DNAP, as in T7 RNAP, the dissociation of the pyrophosphate product breaks the electrostatic link between the catalytic magnesium ions and the basic residues of the fingers subdomain that stabilizes and maintains the closed conformation of the fingers subdomain. ...
Replicative DNA polymerases (DNAPs) move along template DNA in a processive manner. The structural basis of the mechanism of translocation has been better studied in the A-family of polymerases than in the B-family of replicative polymerases. To address this issue, we have determined the X-ray crystal structures of phi29 DNAP, a member of the protein-primed subgroup of the B-family of polymerases, complexed with primer-template DNA in the presence or absence of the incoming nucleoside triphosphate, the pre- and post-translocated states, respectively. Comparison of these structures reveals a mechanism of translocation that appears to be facilitated by the coordinated movement of two conserved tyrosine residues into the insertion site. This differs from the mechanism employed by the A-family polymerases, in which a conserved tyrosine moves into the templating and insertion sites during the translocation step. Polymerases from the two families also interact with downstream single-stranded template DNA in very different ways.
... Consequently, they showed nearly no amplification activity. f29 DBP protein p6 favours the initiation and transition reactions due to a decrease in the K m for the initiating nucleotide, dATP (Blanco et al., 1986(Blanco et al., , 1988, possibly due to a favourable presentation of the templating nucleotide at the polymerization active site (Truniger et al., 2002). Due to the role of residues Phe198 and Lys196 in DNA binding, the templating nucleotide may not be presented in the presence of p6 at the polymerization active sites of mutant DNA polymerases F198V and K196I as favourably as in the wild-type enzyme. ...
For several DNA-dependent DNA polymerases it has been shown that their synthetic and degradative activities are organized in two separated modules. The functional coordination required between them to accomplish successfully the replication process is provided by important contacts with the substrate contributed by residues coming from both modules. These domains are connected by a central "linker" region adjacent to the "YxGG/A" motif, the putative limit of the polymerization domain. We describe here the mutational analysis of phi29 DNA polymerase in several residues of this region, connecting the N- and C-terminal domains and conserved in DNA polymerases able to start replication by protein-priming. The mutant polymerases with the less conservative changes showed reduced DNA binding activity. Additionally, their TP binding capacity was reduced, affecting the TP-deoxynucleotidylation in the absence of template. Interestingly, the role of the residues studied here in DNA binding seems to be especially important to start replication, when the polymerase enters from the closed binary into the ternary complex. These results allow us to propose that this interdomain region of phi29 DNA polymerase is playing an important role for substrate binding including both DNA and TP.
... DNA polymerase was substituted 115 . Substitution of the residue corresponding to Lys-1086 of DNA polymerase ζ resulted in a 500-fold to 5600-fold decrease in the efficiency of nucleotide incorporation for RB69 polymerase 114 , a 60-fold decrease in the efficiency of nucleotide incorporation for human DNA polymerase α 112 , and no detectible DNA polymerase activity for the phage φ29 DNA polymerase 113 . ...
DNA damage blocks replication by classical DNA polymerases, those that replicate nondamaged DNA during normal DNA replication and repair, by altering the geometry of the DNA. Consequently, translesion synthesis, the replication of damaged DNA, is catalyzed by non-classical DNA polymerases, which are capable of accommodating the inherent distorted geometry of damaged DNA. The yeast Rev1 protein (Rev1p) specifically catalyzes the incorporation of cytosine opposite template guanine and several types of DNA damage utilizing a unique mechanism of nucleotide selection whereby the sidechain of Arg-324 acts as the template by forming hydrogen bonds with the incoming cytosine. To better understand the impact of this protein-template-directed mechanism on nucleotide incorporation, I carried out pre-steady-state kinetic studies with Rev1p. Interestingly, I found that Rev1p's specificity for incorporating cytosine is achieved solely at the initial nucleotide-binding step. In this respect, Rev1p differs from all previously investigated DNA polymerases. Based on these findings and on structures of another enzyme, MutM, I suggest possible structures for complexes of Rev1p with the other incoming nucleotides. DNA polymerase ζ, encoded by the REV3 gene, functions in the error-prone replication of a wide range of DNA lesions by extending from nucleotides incorporated opposite template lesions by other polymerases. Here I describe genetic and biochemical studies of five yeast DNA polymerase ζ mutant proteins. Four mutant proteins do not complement the rev3Δ mutation, and these proteins have significantly reduced or no polymerase activity relative to the wild-type protein. However, the K1061A protein partially complements the rev3Δ mutation and has nearly normal polymerase activity. Interestingly, the K1061A protein has increased ability to distinguish between correct and incorrect substrates (increased fidelity and decreased misextension ability). These findings have important implications for the mechanism by which this enzyme accommodates distortions in the DNA caused by mismatches and lesions. Additionally, I genetically characterized 21 mutant proteins, which may also affect the substrate specificity of this enzyme. The P962L, L1054A, T1063A, and G1215A mutant proteins were partially capable of complementing the rev3Δ mutation and are candidates for biochemical characterization, as they may have altered substrate specificity.
Single-molecule technologies can provide detailed information regarding molecular mechanisms and interactions that cannot easily be studied on the bulk scale; generally, individual molecular behaviors cannot be distinguished, and only average characteristics can be measured. Nevertheless, the development of the single-molecule sequencer had a significant impact on conventional in vitro single-molecule research, featuring automated equipment, high-throughput chips, and automated analysis systems. However, the utilization of sequencing technology in in vitro single-molecule research is not yet globally prevalent, owing to the large gap between highly organized single-molecule sequencing and manual-based in vitro single-molecule research. Here, we describe the principles of zero-mode waveguides (ZMWs) and nanopore methods used as single-molecule DNA sequencing techniques, and provide examples of functional biological measurements beyond DNA sequencing that contribute to a global understanding of the current applications of these sequencing technologies. Furthermore, through a comparison of these two technologies, we discuss future applications of DNA sequencing technologies in in vitro single-molecule research.
Fullsize Image
