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High fidelity tRNA aminoacylation by aminoacyl-tRNA synthetases is essential for cell viability. ProXp-ala is a trans-editing protein that is present in all three domains of life and is responsible for hydrolyzing mischarged Ala-tRNAPro and preventing mistranslation of proline codons. Previous studies have shown that, like bacterial prolyl-tRNA synthetase, Caulobacter crescentus ProXp-ala recognizes the unique C1:G72 terminal base pair of the tRNAPro acceptor stem, helping to ensure deacylation of Ala-tRNAPro but not Ala-tRNAAla. The structural basis for C1:G72 recognition by ProXp-ala is still unknown and was investigated here. NMR spectroscopy, binding, and activity assays revealed two conserved residues, K50 and R80, that likely interact with the first base pair, stabilizing the initial protein-RNA encounter complex. Modeling studies are consistent with direct interaction between R80 and the major groove of G72. A third key contact between A76 of tRNAPro and K45 of ProXp-ala was essential for binding and accommodating the CCA-3′ end in the active site. We also demonstrated the essential role that the 2′OH of A76 plays in catalysis. Eukaryotic ProXp-ala proteins recognize the same acceptor stem positions as their bacterial counterparts, albeit with different nucleotide base identities. ProXp-ala is encoded in some human pathogens; thus, these results have the potential to inform new antibiotic drug design.

Russian demographic policy is one of the significant components of its social policy, while over the past six years there has been a negative trend in the demographic situation, accompanied by a sharp decline in birth rates and natural increase. At the same time, state bodies have been actively implementing various measures of demographic policy, using mainly financial incentives, but their arsenal was insufficient. As a hypothesis of the study, we made an assumption about the insufficiently active participation of the media in covering demographic policy measures aimed at stimulating the birth rate, while social and demographic publications are an important component in shaping public opinion. The paper discusses the usage of mass media institutes’ potential as an instrument of demographic policy and support for the family, motherhood and childhood. The authors analyzed the materials posted on the official website of the regional authority responsible for the demographic policy, as well as media publications prepared on these materials. In 2021, almost 27% of publications on the official website of the regional authority responsible for the demographic policy were devoted to family support, motherhood and childhood. On average, there were 14 times more publications in the regional media, which confirms the potential of using this institution to support and promote fertility. Among the most frequent topics of publications were releases on financial measures to support families with children, as well as awarding families with many children.

Aminoacyl-tRNA synthetases (ARSs) catalyze the charging of specific amino acids onto cognate tRNAs, an essential process for protein synthesis. Mutations in ARSs are frequently associated with a variety of human diseases. The human EPRS1 gene encodes a bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) with two catalytic cores and appended domains that contribute to nontranslational functions. In this study, we report compound heterozygous mutations in EPRS1, which lead to amino acid substitutions P14R and E205G in two patients with diabetes and bone diseases. While neither mutation affects tRNA binding or association of EPRS with the multisynthetase complex, E205G in the glutamyl-tRNA synthetase (ERS) region of EPRS is defective in amino acid activation and tRNAGlu charging. The P14R mutation induces a conformational change and altered tRNA charging kinetics in vitro. We propose that the altered catalytic activity and conformational changes in the EPRS variants sensitize patient cells to stress, triggering an increased integrated stress response (ISR) that diminishes cell viability. Indeed, patient-derived cells expressing the compound heterozygous EPRS show heightened induction of the ISR, suggestive of disruptions in protein homeostasis. These results have important implications for understanding ARS-associated human disease mechanisms and development of new therapeutics.

Aminoacyl-tRNA synthetases (aaRS) are ubiquitous enzymes responsible for aminoacyl-tRNA (aa-tRNA) synthesis. Correctly formed aa-tRNAs are necessary for proper decoding of mRNA and accurate protein synthesis. tRNAs possess specific nucleobases that promote selective recognition by cognate aaRSs. Selecting the cognate amino acid can be more challenging because all amino acids share the same peptide backbone and several are isosteric or have similar side chains. Thus, aaRSs can misactivate non-cognate amino acids and produce mischarged aa-tRNAs. If left uncorrected, mischarged aa-tRNAs deliver their non-cognate amino acid to the ribosome resulting in misincorporation into the nascent polypeptide chain. This changes the primary protein sequence and potentially causes misfolding or formation of non-functional proteins that impair cell survival. A variety of proofreading or editing pathways exist to prevent and correct mistakes in aa-tRNA formation. Editing may occur before the amino acid transfer step of aminoacylation via hydrolysis of the aminoacyl-adenylate. Alternatively, post-transfer editing, which occurs after the mischarged aa-tRNA is formed, may be carried out via a distinct editing site on the aaRS where the mischarged aa-tRNA is deacylated. In recent years, it has become clear that most organisms also encode factors that lack aminoacylation activity but resemble aaRS editing domains and function to clear mischarged aa-tRNAs in trans. This review focuses on these trans-editing factors, which are encoded in all three domains of life and function together with editing domains present within aaRSs to ensure that the accuracy of protein synthesis is sufficient for cell survival.

Accurate translation of genetic information into proteins is vital for cell sustainability. ProXp-ala prevents proteome-wide Pro-to-Ala mutations by hydrolyzing misacylated Ala-tRNA(Pro), which is synthesized by prolyl-tRNA synthetase. Bacterial ProXp-ala was previously shown to combine a size-based exclusion mechanism with conformational and chemical selection for the recognition of the alanyl moiety, whereas tRNA(Pro) is selected via recognition of tRNA acceptor-stem elements G72 and A73. The identity of these critical bases changed during evolution with eukaryotic cytosolic tRNA(Pro) possessing a cytosine at the corresponding positions. The mechanism by which eukaryotic ProXp-ala adapted to these changes remains unknown. In this work, recognition of the aminoacyl moiety and tRNA acceptor stem by human (Homo sapiens, or Hs) ProXp-ala was examined. Enzymatic assays revealed that Hs ProXp-ala requires C72 and C73 in the context of Hs cytosolic tRNA(Pro) for efficient deacylation of mischarged Ala-tRNA(Pro). The strong dependence on these bases prevents cross-species deacylation of bacterial Ala-tRNA(Pro) or of Hs mitochondrial Ala-tRNA(Pro) by the human enzyme. Similar to the bacterial enzyme, Hs ProXp-ala showed strong tRNA acceptor-stem recognition but differed in its amino acid specificity profile relative to bacterial ProXp-ala. Changes at conserved residues in both the Hs and bacterial ProXp-ala substrate-binding pockets modulated this specificity. These results illustrate how the mechanism of substrate selection diverged during the evolution of the ProXp-ala family, providing the first example of a trans-editing domain whose specificity evolved to adapt to changes in its tRNA substrate.

Aminoacyl-tRNA synthetases (aaRS) are ubiquitous enzymes responsible for aminoacyl-tRNA (aa-tRNA) synthesis. Correctly formed aa-tRNAs are necessary for proper decoding of mRNA and accurate protein synthesis. tRNAs possess specific nucleobases that promote selective recognition by cognate aaRSs. Selecting the cognate amino acid can be more challenging because all amino acids share the same peptide backbone and several are isosteric or have similar side chains. Thus, aaRSs can misactivate non-cognate amino acids and produce mischarged aa-tRNAs. If left uncorrected, mischarged aa-tRNAs deliver their non-cognate amino acid to the ribosome resulting in misincorporation into the nascent polypeptide chain. This changes the primary protein sequence and potentially causes misfolding or formation of non-functional proteins that impair cell survival. A variety of proofreading or editing pathways exist to prevent and correct mistakes in aa-tRNA formation. Editing may occur before the amino acid transfer step of aminoacylation via hydrolysis of the aminoacyl-adenylate. Alternatively, post-transfer editing, which occurs after the mischarged aa-tRNA is formed, may be carried out via a distinct editing site on the aaRS where the mischarged aa-tRNA is deacylated. In recent years, it has become clear that most organisms also encode factors that lack aminoacylation activity but resemble aaRS editing domains and function to clear mischarged aa-tRNAs in trans. This review focuses on these trans-editing factors, which are encoded in all three domains of life and function together with editing domains present within aaRSs to ensure that the accuracy of protein synthesis is sufficient for cell survival.

Aminoacyl-tRNA synthetases catalyze the attachment of cognate amino acids onto tRNAs. To avoid mistranslation, editing mechanisms evolved to maintain tRNA aminoacylation fidelity. For instance, while rejecting the majority of non-cognate amino acids via discrimination in the synthetic active site, prolyl-tRNA synthetase (ProRS) misactivates and mischarges Ala and Cys, which are similar in size to cognate Pro. Ala-tRNAPro is specifically hydrolyzed by the editing domain of ProRS in cis, while YbaK, a free-standing editing domain, clears Cys-tRNAPro in trans. ProXp-ala is another editing domain that clears Ala-tRNAPro in trans. YbaK does not appear to possess tRNA specificity, readily deacylating Cys-tRNACysin vitro. We hypothesize that YbaK binds to ProRS to gain specificity for Cys-tRNAPro and avoid deacylation of Cys-tRNACys in the cell. Here, in vivo evidence for ProRS-YbaK interaction was obtained using a split-green fluorescent protein assay. Analytical ultracentrifugation and native mass spectrometry were used to investigate binary and ternary complex formation between ProRS, YbaK, and tRNAPro. Our combined results support the hypothesis that the specificity of YbaK toward Cys-tRNAPro is determined by the formation of a three-component complex with ProRS and tRNAPro and establish the stoichiometry of a ‘triple-sieve’ editing complex for the first time.

Molecular sieves ensure proper pairing of tRNAs and amino acids during aminoacyl-tRNA biosynthesis, thereby avoiding detrimental effects of mistranslation on cell growth and viability. Mischarging errors are often corrected through the activity of specialized editing domains present in some aminoacyl-tRNA synthetases or via single-domain

Molecular sieves ensure proper pairing of tRNAs and amino acids during aminoacyl-tRNA biosynthesis, thereby avoiding detrimental effects of mistranslation on cell growth and viability. Mischarging errors are often corrected through the activity of specialized editing domains present in some aminoacyl-tRNA synthetases or via single-domain trans-editing proteins. ProXp-ala is a ubiquitous trans-editing enzyme that edits Ala-tRNAPro, the product of Ala mischarging by prolyl-tRNA synthetase, although the structural basis for discrimination between correctly charged Pro-tRNAPro and mischarged Ala-tRNAAla is unclear. Deacylation assays using substrate analogs reveal that size discrimination is only one component of selectivity. We used NMR spectroscopy and sequence conservation to guide extensive site-directed mutagenesis of Caulobacter crescentus ProXp-ala, along with binding and deacylation assays to map specificity determinants. Chemical shift perturbations induced by an uncharged tRNAPro acceptor stem mimic, microhelixPro, or a nonhydrolyzable mischarged Ala-microhelixPro substrate analog identified residues important for binding and deacylation. Backbone 15N NMR relaxation experiments revealed dynamics for a helix flanking the substrate binding site in free ProXp-ala, likely reflecting sampling of open and closed conformations. Dynamics persist on binding to the uncharged microhelix, but are attenuated when the stably mischarged analog is bound. Computational docking and molecular dynamics simulations provide structural context for these findings and predict a role for the substrate primary α-amine group in substrate recognition. Overall, our results illuminate strategies used by a trans-editing domain to ensure acceptance of only mischarged Ala-tRNAPro, including conformational selection by a dynamic helix, size-based exclusion, and optimal positioning of substrate chemical groups.

Polymerase fidelity, at its most basic level, is determined by the free energy difference between the highest energy barriers along the correct and incorrect dNTP1 incorporation pathways. Since fidelity requires such a comparison, an ever-increasing gap between the structural and mechanistic information available for matched dNTP incorporation compared with that concerning mismatched dNTP incorporation renders the molecular mechanism of DNA polymerase fidelity unclear (1,2). The thermodynamic instability inherent to polymerase mismatched ternary complexes makes them difficult to crystallize. For this reason, we do not yet have available a structure representing a truly functional prechemistry closed mismatched ternary complex (E′DnN, Scheme 1), although complexes depicting mispairs within the active site (3,4) and mismatch extension (5,6) for various polymerases have been resolved. Likewise, kinetic data for mismatched dNTP incorporations by many DNA polymerases have been reported (7−13), yet commonly used kinetic approaches prevent characterization of individual steps in the mismatched dNTP incorporation pathway. Scheme 1 The Ile260 residue of Pol β is located in the hydrophobic hinge region between the palm and the C-terminal fingers subdomain (14,15). Several Pol β variants with alterations in residues lining this hinge region have demonstrated compromised fidelity (16−20). The mutator activity of I260Q was first identified by a genetic screen (21), and subsequent kinetic characterization showed that while this mutant possessed parameters of DNA binding similar to that of wild-type, it interestingly showed loose binding discrimination of mismatched dNTP substrates, and thus a low fidelity (22). These characteristics make the I260Q mutant of specific interest in fidelity studies of Pol β, in order to further understand how one mutation can so drastically alter the mismatch discrimination profile of this enzyme. Stopped-flow analyses of Pol β, using either 2-aminopurine (2-AP), a fluorescent analogue of adenine and guanine, or Pol βʼs sole Trp325 residue, have been very important in advancing Pol β mechanism studies (23−26). Both probes serve as independent reporters of global enzyme−DNA conformational changes which occur during the catalysis of dNTP incorporation into DNA. While monitoring the fluorescence emission of either 2-AP or Trp325 in stopped-flow, the resultant trace observed during correct dNTP incorporation possesses two phases of fluorescence change. Intricate studies involving a variety of chemical probes have been conducted in order to determine the nature of these two phases. The collective findings of these studies support a model in which the fast fluorescence transition is attributed to dNTP-induced subdomain conformational change (step 2, Scheme 1) to form the ternary closed complex prior to chemistry (step 4, Scheme 1), and the slow fluorescence transition is attributed to subdomain reopening (step 6, Scheme 1) after the rate-limiting chemical step. This work aims to apply our previously established stopped-flow fluorescence methods, as well as steady-state fluorescence spectroscopy to explore two specific aspects of polymerase fidelity: (i) the mechanism by which WT Pol β discriminates against incorrect dNTP substrate to maintain a moderately high fidelity, and (ii) the deviation of this mechanism in generation of a lower fidelity variant by a single I260Q mutation. Such topics are significant in developing a full understanding of the unique means by which DNA polymerases are able to choose a correct dNTP substrate, whose identity is ever-changing with each round of catalysis, from a pool four nucleotides. Moreover, while it is interesting to comprehend how a polymerase accomplishes this feat to achieve a certain fidelity in DNA replication, it is equally interesting to investigate how a single residue mutation can alter its fidelity. Here, we focus on the kinetic pathway of misincorporation, while another recent paper using small-angle X-ray scattering (SAXS) and molecular modeling lends complementary insights into structural aspects of misincorporation (27). The results of our analyses suggest that Pol β incorporates both correct and mismatched dNTP via analogous kinetic mechanisms differing at the transition state of the chemical step. Furthermore, the divergence in fidelity between WT and I260Q is consequent of differences in ability to stabilize the mismatched ternary complex and chemical transition state.

A sequential ordered substrate binding established previously for several DNA polymerases is generally extended to all DNA polymerases, and the characterization of novel polymerases is often based on the assumption that the enzymes should productively bind DNA substrate first, followed by template-directed dNTP binding. The comprehensive kinetic study of DNA polymerase X (Pol X) from African swine fever virus reported here is the first analysis of the substrate binding order performed for a low-fidelity DNA polymerase. A classical steady-state kinetic approach using substrate analogue inhibition assays demonstrates that Pol X does not follow the bi-bi ordered mechanism established for other DNA polymerases. Further, using isotope-trapping experiments and stopped-flow fluorescence assays, we show that Pol X can bind Mg (2+).dNTPs in a productive manner in the absence of DNA substrate. We also show that DNA binding to Pol X, although rapid, may not always be productive. Furthermore, we show that binding of Mg (2+).dNTP to Pol X facilitates subsequent formation of the catalytically competent Pol X.DNA.dNTP ternary complex, whereas DNA binding prior to dNTP binding brings the enzyme into a nonproductive conformation where subsequent nucleotide substrate binding is hindered. Together, our results suggest that Pol X prefers an ordered sequential mechanism with Mg (2+).dNTP as the first substrate.

30 p.-14 fig. Zhao, Huaying, et alt., Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Presented here is the development a semi-rational protein engineering approach that uses information from protein structure coupled with established DNA manipulation techniques to design and create multiple crossover libraries from non-homologous genes. The utility of structure-based combinatorial protein engineering (SCOPE) was demonstrated by its application to two distantly related members of the X-family of DNA polymerases: rat DNA polymerase beta (Pol beta) and African swine fever virus DNA polymerase X (Pol X). These proteins share similar folds but have low sequence identity, and differ greatly in both size and activity. "Equivalent" subdomain elements of structure were designed on the basis of the tertiary structure of Pol beta and the corresponding regions of Pol X were inferred from homology modeling and sequence alignment analysis. Libraries of chimeric genes with up to five crossovers were synthesized in a series of PCR reactions by employing hybrid oligonucleotides that code for variable connections between structural elements. Genetic complementation in Escherichia coli enabled identification of several novel DNA polymerases with enhanced phenotypes. Both the composition of structural elements and the manner in which they were linked were shown to be essential for this property, indicating the importance of these aspects of design.

DNA polymerases slide on DNA during replication, and the interface must be mobile for various conformational changes. The role of lubricant interfacial water is not understood. In this report, we systematically characterize the water dynamics at the interface and in the active site of a tight binding polymerase (pol β) in its binary complex and ternary state using tryptophan as a local optical probe. Using femtosecond spectroscopy, we observed that upon DNA recognition the surface hydration water is significantly confined and becomes bound water at the interface, but the dynamics are still ultrafast and occur on the picosecond time scale. These interfacial water molecules are not trapped but are mobile in the heterogeneous binding nanospace. Combining our findings with our previous observation of ultrafast water motions at the interface of a loose binding polymerase (Dpo4), we conclude that the binding interface is dynamic and the water molecules in various binding clefts, channels, and caves are mobile and even fluid with different levels of mobility for loose or tight binding polymerases. Such a dynamic interface should be general to all DNA polymerase complexes to ensure the biological function of DNA synthesis.

To obtain a high level of accuracy during protein synthesis, several different quality control steps are employed by the cellular machinery. The aminoacyl-tRNA synthetases (aaRS) play a critical role in identifying amino acids and pairing them with their cognate tRNAs. Prolyl-tRNA synthetase (ProRS) from all three domains of life has been shown to mischarge alanine and cysteine onto tRNAPro. Most bacterial ProRSs have an editing domain that deacylates mischarged Ala-tRNAPro. However, this double-sieve editing mechanism is not sufficient to clear Cys-tRNAPro. Instead, a free-standing homolog of the ProRS editing domain called YbaK deacylates mischarged Cys-tRNAPro species.We have demonstrated that tRNAPro, ProRS and YbaK form a ternary complex in vitro and in vivo, but the details of this complex are not known.Based on preliminary computational studies, we hypothesize that the alanine editing domain of ProRS undergoes a conformational change to facilitate YbaK binding. In addition, the CCA-3′ end of the tRNA must also be involved in significant conformational changes, translocating between the synthetic active site, the ProRS editing domain active site located 35 A away, and YbaK. To probe these protein and RNA domain movements, we devised a fluorescence resonance energy transfer (FRET)-based approach. To date, using ensemble time-resolved FRET, we have measured an ∼20 A conformational change in the ProRS editing domain upon YbaK binding, confirming our hypothesis of a large conformational change to accommodate YbaK. Moreover, the distance between tRNA and YbaK differ by ∼15 A in the presence and absence of ProRS, further verifying a conformational change. Current studies are aimed at obtaining additional distance constraints between components of the ternary complex.

Investigation into the mechanistic origin of the natural divergence of fidelity among DNA polymerases has been a subject of study for several decades. This work aims to provide an overview of the variety of approaches that have been used to examine the catalytic mechanism of DNA polymerases, with a large focus on the use of DNA polymerase β (Pol β) as a model system. The chapter encompasses the contributions of structural, kinetic, and computational studies in the advancement of our overall understanding of DNA polymerase catalysis.

A complete understanding of the kinetic mechanism of fidelity requires comparison of correct and incorrect dNTP incorporation pathways in both the forward and reverse directions. The studies presented here focus on the dNTP-induced conformational step, which has historically been proposed by many to be the major determinant of fidelity. As it was recently highlighted [Tsai, Y. C., and Johnson, K. A. (2006) Biochemistry 45, 9675-9687], chemistry can be the slowest step in the forward direction of the correct dNTP incorporation pathway, yet the corresponding microscopic rate constant would not contribute toward fidelity in the case when the reverse rate of the conformational step is slower than chemistry. Here we use a stopped-flow technique to directly measure the reverse rate of the conformational step in the DNA polymerase beta (Pol beta) kinetic pathway. Extensive pre-steady-state kinetic studies presented include the utilization of 2-aminopurine-labeled DNA substrates, 2-aminopurine nucleotide triphosphate, a nonhydrolyzable nucleotide analogue dAMPCPP, and a rapid sequential mixing reaction scheme. Additionally, the effect of mismatched dNTPs, various metal ions, and the presence of the 3'-terminal hydroxyl group of the primer on the rate of the reverse "opening" conformational step were analyzed. Our analyses indicate that reverse "opening" is drastically facilitated in the presence of mismatched ternary complexes, which is in agreement with the hypothesis that the ternary complex is destabilized by the presence of incorrect dNTP. By analysis of the relative magnitudes of chemistry and reverse "opening" in the presence of both matched and mismatched matched ternary complexes, this work further validates that, for Pol beta, fidelity is dictated by the differences in free energy required to reach the highest energy transition state of the chemical step.

After extensive studies spanning over half a century, there is little consensus on the kinetic mechanism of DNA polymerases. Using stopped-flow fluorescence assays for mammalian DNA polymerase beta (Pol beta), we have previously identified a fast fluorescence transition corresponding to conformational closing, and a slow fluorescence transition matching the rate of single-nucleotide incorporation. Here, by varying pH and buffer viscosity, we have decoupled the rate of single-nucleotide incorporation from the rate of the slow fluorescence transition, thus confirming our previous hypothesis that this transition represents a conformational event after chemistry, likely subdomain reopening. Analysis of an R258A mutant indicates that rotation of the Arg258 side chain is not rate-limiting in the overall kinetic pathway of Pol beta, yet is kinetically significant in subdomain reopening. We have extended our kinetic analyses to a high-fidelity polymerase, Klenow fragment (KF), and a low-fidelity polymerase, African swine fever virus DNA polymerase X (Pol X), and showed that they follow the same kinetic mechanism as Pol beta, while differing in relative rates of single-nucleotide incorporation and the putative conformational reopening. Our data suggest that the kinetic mechanism of Pol beta is not an exception among polymerases, and furthermore, its delineated kinetic mechanism lends itself as a platform for comparison of the kinetic properties of different DNA polymerases and their mutants.

The kinetic mechanism and the structural bases of the fidelity of DNA polymerases are still highly controversial. Here we report the use of three probes in the stopped-flow studies of Pol beta to obtain new, direct evidence for our previous interpretations: (a) Increasing the viscosity of the reaction buffer by sucrose or glycerol is expected to slow down the conformational change differentially, and it was shown to slow down the first (fast) fluorescence transition selectively. (b) Use of dNTPalphaS in place of dNTP is expected to slow down the chemical step preferentially, and it was shown to slow down the second (slow) fluorescence transition selectively. (c) The substitution-inert Rh(III)dNTP was used to show for the first time that the slow fluorescence change occurs after mixing of Pol beta.DNA.Rh(III)dNTP with Mg(II). These results, along with crystal structures, suggest that the subdomain-closing conformational change occurs before binding of the catalytic Mg(II) while the rate-limiting step occurs after binding of the catalytic Mg(II). These results provide new evidence to the mechanism we suggested previously, but do not support the results of three recent papers of computational studies. The results were further supported by a "sequential mixing" stopped-flow experiment that used no analogues, and thus ruled out the possibility that the discrepancy between experimental and computational results is due to the use of analogues. The methodologies can be used to examine other DNA polymerases to answer whether the properties of Pol beta are exceptional or general.