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Phage RB69 B-family DNA polymerase is responsible for the overall high fidelity of RB69 DNA synthesis. Fidelity is compromised when conserved Tyr567, one of the residues that form the nascent polymerase base-pair binding pocket, is replaced by alanine. The Y567A mutator mutant has an enlarged binding pocket and can incorporate and extend mispairs e...
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... The roster of mutational classes showed a significant decrease in T → C transitions with the double replacement compared to that with Pol Y567A alone. There were at least five sequence contexts where T → C mutations either were absent or appeared only once in the double-mutant spectrum but were strongly represented in the single-mutant spectrum (Fig. 2a). Overall, base substitution rates decreased by 14-fold in the double mutant, with the decrease in T·dGMP rates being as much as 35-fold compared to the Pol Y567A polymerase ( Table 5). The other transition mutation rates decreased 7-fold for C·dATP, 8-fold for G·dTMP, and 5-fold for A·dCMP. Transversion mutations were less frequent in ...
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... over all detectable sites in the lacZα template and for Exo + and Exo − single and double Pol mutants are presented in Table 5. In Exo − background, the spectra for the single and double mutants showed no striking differences, and the distribution of mutations along the lacZα sequence revealed no significant differences between the two enzymes ( Fig. 2b and c). 16 The number of detectable sites is not defined for deletions N1 nt, and their mutation rates are therefore not normalized to the number of opportunities and thus only appear to be larger than other normalized ...
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... mutant would be increased exonuclease activity. We therefore conducted exonuclease assays on substrates with either a correct base pair or a T·dGMP mismatch at the 3′ terminus to compare the various polymerase variants. However, both Pol S565G and Pol S565G/Y567A polymerases were less efficient than the wild-type and Pol Y567A polymerases in Fig. 2. lacZα mutational spectra. The 5′ → 3′ sequence of the viral template strand of the lacZα sequence in M13mp2 is shown from position −84 through position +197, where +1 is the first transcribed base and every 10th base is underlined. Upper-case letters indicate base substitutions. The deletion of a single base is indicated by "Δ," ...
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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...
Citations
... Despite the information learned from structural and biochemical studies of DNA polymerases, Jan concluded that fidelities of wild-type and mutant RB69 DNA polymerases determined in vitro only partially reflected their fidelities in vivo (Bebenek et al. 2002). For example, biochemical studies of engineered RB69 DNA polymerases showed that increasing the size of the nucleotide-binding pocket reduced nucleotide insertion fidelity, but that was countered by in vivo mutation studies (Trzemecka et al. 2010) that forced reconsideration of the biochemical studies (Xia et al. 2011). Hence, there is a real and continuing need for in vivo replication fidelity experiments. ...
John W. Drake died 02-02-2020, a mathematical palindrome, which he would have enjoyed, given his love of “word play and logic,” as stated in his obituary and echoed by his family, friends, students, and colleagues. Many aspects of Jan’s career have been reviewed previously, including his early years as a Caltech graduate student, and when he was editor-in-chief, with the devoted assistance of his wife Pam, of this journal for 15 impactful years. During his editorship, he raised the profile of GENETICS as the flagship journal of the Genetics Society of America and inspired and contributed to the creation of the Perspectives column, coedited by Jim Crow and William Dove. At the same time, Jan was building from scratch the Laboratory of Molecular Genetics on the newly established Research Triangle Park campus of the National Institute of Environmental Health Science, which he headed for 30 years. This commentary offers a unique perspective on Jan’s legacy; we showcase Jan’s 1969 benchmark discovery of antimutagenic T4 DNA polymerases and the research by three generations (and counting) of scientists whose research stems from that groundbreaking discovery. This is followed by a brief discussion of Jan’s passion: his overriding interest in analyzing mutation rates across species. Several anecdotal stories are included to bring alive one of Jan’s favorite phrases, “to think like a geneticist.” We feature Jan’s genetical approach to mutation studies, along with the biochemistry of DNA polymerase function, our area of expertise. But in the end, we acknowledge, as Jan did, that genetics, also known as in vivo biochemistry, prevails.
... Several studies were conducted to investigate the effects of amino acid exchanges on polymerase fidelity through mismatch extension selectivity of DNA polymerases from sequence family A. In E. coli DNA polymerase I and Thermus aquaticus DNA polymerase it has been shown that within motif C, which plays a key role in primer/template recognition [42], hydrophobic amino acid substitutions result in polymerase variants with increased mismatch extension selectivity [35,43,44]. Amino acid exchanges have been analyzed for the B-family DNA polymerase RB69 and the eukaryotic DNA polymerase δ in comparison to A-family members, showing furthermore that no direct comparisons can be made between analogous amino acids even within the same sequence family: exchanges that led to antimutator variants of Pol δ, turned the corresponding RB69 mutant into a strong mutator [45]. Although B family polymerases are used in many core biotechnological applications, they are still significantly less well understood and especially structural studies of the ternary complex remain elusive [46][47][48]. ...
Fidelity and selectivity of DNA polymerases are critical determinants for the biology of life, as well as important tools for biotechnological applications. DNA polymerases catalyze the formation of DNA strands by adding deoxynucleotides to a primer, which is complementarily bound to a template. To ensure the integrity of the genome, DNA polymerases select the correct nucleotide and further extend the nascent DNA strand. Thus, DNA polymerase fidelity is pivotal for ensuring that cells can replicate their genome with minimal error. DNA polymerases are, however, further optimized for more specific biotechnological or diagnostic applications. Here we report on the semi-rational design of mutant libraries derived by saturation mutagenesis at single sites of a 3’-5’-exonuclease deficient variant of Thermococcus kodakaraensis DNA polymerase (KOD pol) and the discovery for variants with enhanced mismatch extension selectivity by screening. Sites of potential interest for saturation mutagenesis were selected by their proximity to primer or template strands. The resulting libraries were screened via quantitative real-time PCR. We identified three variants with single amino acid exchanges—R501C, R606Q, and R606W—which exhibited increased mismatch extension selectivity. These variants were further characterized towards their potential in mismatch discrimination. Additionally, the identified enzymes were also able to differentiate between cytosine and 5-methylcytosine. Our results demonstrate the potential in characterizing and developing DNA polymerases for specific PCR based applications in DNA biotechnology and diagnostics.
... Here the most important mutants are discussed in light of the mechanisms proposed in this study for translocation and active site switching, which involve Tyr567 located at the center of the back wall between Tyr416 and Gly568 in the base pair binding pocket (Supplementary Figure S4d-f). Substitution for this residue with Ala substantially increases the error rate and also leads to other puzzling features (61,62). The available structures of Y567A mutant (e.g. ...
... Therefore the overall structural effect of Y567A is a smaller, rather than larger (63), binding pocket except that the back wall is no longer intact. Compared to the wild-type, Y567A mutant has reduced affinity to bind normal DNA duplex but elevated affinity to bind DNA duplex with mismatched base pair (62). Similar results also show that this substitution increases binding affinity of dGTP by 45-fold opposite a template G compared to the wild-type (64). ...
DNA polymerases in family B are workhorses of DNA replication that carry out the bulk of the job at a high speed with high accuracy. A polymerase in this family relies on a built-in exonuclease for proofreading. It has not been observed at the atomic resolution how the polymerase advances one nucleotide space on the DNA template strand after a correct nucleotide is incorporated, that is, a process known as translocation. It is even more puzzling how translocation is avoided after the primer strand is excised by the exonuclease and returned back to the polymerase active site once an error occurs. The structural events along the bifurcate pathways of translocation and proofreading have been unwittingly captured by hundreds of structures in Protein Data Bank. This study analyzes all available structures of a representative member in family B and reveals the orchestrated event sequence during translocation and proofreading.
... Numerous biochemical and structural studies have focused on the active site of RB69 DNA Pol and provided information about the function of several highly conserved residues [9][10][11][12][13][14]. These include immediate neighbors of the catalytic aspartate triad D411, D621 and D623, engaged directly or indirectly in binding the divalent metal ion and the 3′-end of the primer, as well as several fingers subdomain residues constituting the dNTP binding site and responsible for mismatch discrimination. ...
... The observed antimutator phenotype of D714A polymerase in vitro may result from increased exonuclease activity and/or decreased ability to form binary polymerase-DNA complexes [11,16]. Thus, we decided to test whether the D714A substitution might decrease the DNA binding affinity of the polymerase and/or change its ability to exist in the editing mode required for efficient proofreading. ...
Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A), that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol(D714A) mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.
... Pol δ Y708A of motif B affects the same surface, contacting the minor-grove side of the base. The analogous mutant in RB69 Pol (Y567A) is a strong mutator, but this phenotype is suppressed by a second substitution in the dNTP binding site, S565G (Trzemecka et al., 2010;Xia et al., 2011). Structural and biochemical analyses show that G565 in the S565G,Y567A double mutant limits the flexibility of the template base position (Xia et al., 2011) and that, under certain conditions in vitro, the double-mutant polymerase dissociates more rapidly from duplexes with mispaired primer termini (Trzemecka et al., 2010). ...
... The analogous mutant in RB69 Pol (Y567A) is a strong mutator, but this phenotype is suppressed by a second substitution in the dNTP binding site, S565G (Trzemecka et al., 2010;Xia et al., 2011). Structural and biochemical analyses show that G565 in the S565G,Y567A double mutant limits the flexibility of the template base position (Xia et al., 2011) and that, under certain conditions in vitro, the double-mutant polymerase dissociates more rapidly from duplexes with mispaired primer termini (Trzemecka et al., 2010). Thus, changes to the binding pocket may influence not only nucleotide selectivity, but also stability of the replication complex. ...
Evolution balances DNA replication speed and accuracy to optimize replicative fitness and genetic stability. There is no selective pressure to improve DNA replication fidelity beyond the background mutation rate from other sources, such as DNA damage. However, DNA polymerases remain amenable to amino acid substitutions that lower intrinsic error rates. Here, we review these 'antimutagenic' changes in DNA polymerases and discuss what they reveal about mechanisms of replication fidelity. Pioneering studies with bacteriophage T4 DNA polymerase (T4 Pol) established the paradigm that antimutator amino acid substitutions reduce replication errors by increasing proofreading efficiency at the expense of polymerase processivity. The discoveries of antimutator substitutions in proofreading-deficient 'mutator' derivatives of bacterial Pols I and III and yeast Pol δ suggest there must be additional antimutagenic mechanisms. Remarkably, many of the affected amino acid positions from Pol I, Pol III, and Pol δ are similar to the original T4 Pol substitutions. The locations of antimutator substitutions within DNA polymerase structures suggest that they may increase nucleotide selectivity and/or promote dissociation of primer termini from polymerases poised for misincorporation, leading to expulsion of incorrect nucleotides. If misincorporation occurs, enhanced primer dissociation from polymerase domains may improve proofreading in cis by an intrinsic exonuclease or in trans by alternate cellular proofreading activities. Together, these studies reveal that natural selection can readily restore replication error rates to sustainable levels following an adaptive mutator phenotype.
Given the increasing complexity of simulated molecular systems, and the fact that simulation times have now reached milliseconds to seconds, immense amounts of data (in the gigabyte to terabyte range) are produced in current molecular dynamics simulations. Manual analysis of these data is a very time-consuming task, and important events that lead from one intermediate structure to another can become occluded in the noise resulting from random thermal fluctuations. To overcome these problems and facilitate a semi-automated data analysis, we introduce in this work a measure based on C(α) torsion angles: torsion angles formed by four consecutive C(α) atoms. This measure describes changes in the backbones of large systems on a residual length scale (i.e., a small number of residues at a time). Cluster analysis of individual C(α) torsion angles and its fuzzification led to continuous time patches representing (meta)stable conformations and to the identification of events acting as transitions between these conformations. The importance of a change in torsion angle to structural integrity is assessed by comparing this change to the average fluctuations in the same torsion angle over the complete simulation. Using this novel measure in combination with other measures such as the root mean square deviation (RMSD) and time series of distance measures, we performed an in-depth analysis of a simulation of the open form of DNA polymerase I. The times at which major conformational changes occur and the most important parts of the molecule and their interrelations were pinpointed in this analysis. The simultaneous determination of the time points and localizations of major events is a significant advantage of the new bottom-up approach presented here, as compared to many other (top-down) approaches in which only the similarity of the complete structure is analyzed.
Accurate DNA synthesis is vital to maintain genome stability and ensure propagation of species. Synthetic errors have far reaching consequences. Therefore, DNA synthesis is remarkably accurate. The high fidelity is mainly achieved through three steps: ① nucleotide selection, which is based on hydrogen, base pair shape, or some other elements; ② 3'→5' exonuclease proofreading, which removes mis-incorporated nucleotides in cis or trans; ③ repair process, which could correct mismatched nucleotides escaping from proofreading, such as mismatch repair, excission repair, homologous recombination repair, and translesion DNA synthesis. Because all polymerases are suitable targets for anticancer or antiviral drugs, their fidelity is involved in drug resistance and side effects. Understanding the molecular basis of synthesis fidelity is of vital importance. In this review, the fidelity mechanisms of DNA synthesis will be discussed in detail. Furthermore, their application perspective was discussed.
Like most phages with double-stranded DNA, phage T4 exits the infected host cell by a lytic process requiring, at a minimum,
an endolysin and a holin. Unlike most phages, T4 can sense superinfection (which signals the depletion of uninfected host
cells) and responds by delaying lysis and achieving an order-of-magnitude increase in burst size using a mechanism called
lysis inhibition (LIN). T4 r mutants, which are unable to conduct LIN, produce distinctly large, sharp-edged plaques. The discovery of r mutants was key to the foundations of molecular biology, in particular to discovering and characterizing genetic recombination
in T4, to redefining the nature of the gene, and to exploring the mutation process at the nucleotide level of resolution.
A number of r genes have been described in the past 7 decades with various degrees of clarity. Here we describe an extensive and perhaps
saturating search for T4 r genes and relate the corresponding mutational spectra to the often imperfectly known physiologies of the proteins encoded
by these genes. Focusing on r genes whose mutant phenotypes are largely independent of the host cell, the genes are rI (which seems to sense superinfection and signal the holin to delay lysis), rIII (of poorly defined function), rIV (same as sp and also of poorly defined function), and rV (same as t, the holin gene). We did not identify any mutations that might correspond to a putative rVI gene, and we did not focus on the famous rII genes because they appear to affect lysis only indirectly.
The contents of the plenary lectures presented at the Plant and Animal Genome (PAG) meeting in January 2011 are summarized in order to provide some insights into the advances in plant, animal and microbe genome studies as they impact on our understanding of complex biological systems. The areas of biology covered include the dynamics of genome change, biological recognition processes and the new processes that underpin investment in science. This overview does not attempt to summarize the diversity of activities that are covered during the PAG through workshops, posters and the suppliers of cutting-edge technologies, but reviews major advances in specific research areas.
We have previously observed that stepwise replacement of amino acid residues in the nascent base-pair binding pocket of RB69 DNA polymerase (RB69pol) with Ala or Gly expanded the space in this pocket, resulting in a progressive increase in misincorporation. However, in vivo results with similar RB69pol nascent base-pair binding pocket mutants showed that mutation rates, as determined by the T4 phage rI forward assay and rII reversion assay, were significantly lower for the RB69pol S565G/Y567A double mutant than for the Y567A single mutant, the opposite of what we would have predicted. To investigate the reasons for this unexpected result, we have determined the pre-steady-state kinetic parameters and crystal structures of relevant ternary complexes. We found that the S565G/Y567A mutant generally had greater base selectivity than the Y567A mutant and that the kinetic parameters for dNMP insertion, excision of the 3'-terminal nucleotide residue, and primer extension beyond a mispair differed not only between these two mutants but also between the two highly mutable sequences in the T4 rI complementary strand. Comparison of the crystal structures of these two mutants with correct and incorrect incoming dNTPs provides insight into the unexpected increase in the fidelity of the S565G/Y567A double mutant. Taken together, the kinetic and structural results provide a basis for integrating and interpreting in vivo and in vitro observations.
