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BBA - Proteins and Proteomics (v.1804, #5)

Editorial Board (pp. i).

DNA polymerases: Perfect enzymes for an imperfect world by Anthony J. Berdis Guest Editor (pp. 1029-1031).

This Special Thematic Issue explores the molecular properties of DNA polymerases as extraordinary biological catalysts. In this short introductory chapter, I briefly highlight some of the most important concepts from the articles contained within this Special Issue. The contents of this Special Issue are arranged into distinct sub-categories corresponding to mechanistic studies of faithful DNA polymerization, studies of "specialized" DNA polymerases that function on damaged DNA, and DNA polymerases that are of therapeutic importance against various diseases. Emphasis is placed on understanding the dynamic cellular roles and biochemical functions of DNA polymerases, and how their structure and mechanism impact their cellular roles.

Keywords: DNA polymerases; Replication; Repair; Translesion DNA synthesis; Mutagenesis

Techniques used to study the DNA polymerase reaction pathway by Catherine M. Joyce (pp. 1032-1040).

A minimal reaction pathway for DNA polymerases was established over 20years ago using chemical-quench methods. Since that time there has been considerable interest in noncovalent steps in the reaction pathway, conformational changes involving the polymerase or its DNA substrate that may play a role in substrate specificity. Fluorescence-based assays have been devised in order to study these conformational transitions and the results obtained have added new detail to the reaction pathway.

Keywords: DNA polymerase; Reaction pathway; Kinetics; Fluorescence

The kinetic and chemical mechanism of high-fidelity DNA polymerases by Kenneth A. Johnson (pp. 1041-1048).

This review summarizes our current understanding of the structural, kinetic and thermodynamic basis for the extraordinary accuracy of high-fidelity DNA polymerases. High-fidelity DNA polymerases, such as the enzyme responsible for the replication of bacteriophage T7 DNA, discriminate against similar substrates with an accuracy that approaches one error in a million base pairs while copying DNA at a rate of approximately 300 base pairs per second. When the polymerase does make an error, it stalls, giving time for the slower proofreading exonuclease to remove the mismatch so that the overall error frequency approaches one in a billion. Structural analysis reveals a large change in conformation after nucleotide binding from an open to a closed state. Kinetic analysis has shown that the substrate-induced structural change plays a key role in the discrimination between correct and incorrect base pairs by governing whether a nucleotide will be retained and incorporated or rapidly released.

Keywords: DNA polymerase; Mechanism; Kinetics; Transient; Stopped-flow; Fluorescence; Quench-flow; Conformational change; Enzyme dynamics

DNA polymerase proofreading: Multiple roles maintain genome stability by Linda J. Reha-Krantz (pp. 1049-1063).

DNA polymerase proofreading is a spell-checking activity that enables DNA polymerases to remove newly made nucleotide incorporation errors from the primer terminus before further primer extension and also prevents translesion synthesis. DNA polymerase proofreading improves replication fidelity ∼100-fold, which is required by many organisms to prevent unacceptably high, life threatening mutation loads. DNA polymerase proofreading has been studied by geneticists and biochemists for >35 years. A historical perspective and the basic features of DNA polymerase proofreading are described here, but the goal of this review is to present recent advances in the elucidation of the proofreading pathway and to describe roles of DNA polymerase proofreading beyond mismatch correction that are also important for maintaining genome stability.

Keywords: DNA polymerase proofreading; DNA replication fidelity; Mutator and antimutator DNA polymerases; Proofreading pathway; Active site switching; Okazaki fragment maturation

Non-natural nucleotides as probes for the mechanism and fidelity of DNA polymerases by Irene Lee; Anthony J. Berdis (pp. 1064-1080).

DNA is a remarkable macromolecule that functions primarily as the carrier of the genetic information of organisms ranging from viruses to bacteria to eukaryotes. The ability of DNA polymerases to efficiently and accurately replicate genetic material represents one of the most fundamental yet complex biological processes found in nature. The central dogma of DNA polymerization is that the efficiency and fidelity of this biological process is dependent upon proper hydrogen-bonding interactions between an incoming nucleotide and its templating partner. However, the foundation of this dogma has been recently challenged by the demonstration that DNA polymerases can effectively and, in some cases, selectively incorporate non-natural nucleotides lacking classic hydrogen-bonding capabilities into DNA. In this review, we describe the results of several laboratories that have employed a variety of non-natural nucleotide analogs to decipher the molecular mechanism of DNA polymerization. The use of various non-natural nucleotides has lead to the development of several different models that can explain how efficient DNA synthesis can occur in the absence of hydrogen-bonding interactions. These models include the influence of steric fit and shape complementarity, hydrophobicity and solvation energies, base-stacking capabilities, and negative selection as alternatives to rules invoking simple recognition of hydrogen-bonding patterns. Discussions are also provided regarding how the kinetics of primer extension and exonuclease proofreading activities associated with high-fidelity DNA polymerases are influenced by the absence of hydrogen-bonding functional groups exhibited by non-natural nucleotides.

Keywords: Abbreviations; dNTP; deoxynucleoside triphosphate; A; adenine; C; cytidine; G; guanine; T; thymine; dAMP; adenosine-2′-deoxyriboside monophosphate; dCMP; cytosine-2′-deoxyriboside; dGMP; guanosine-2′-deoxyriboside dTMP, thymine-2′-deoxyriboside; dF; 2,4-difluorotoluene; dFMP; 2,4-difluorotoluene monophosphate; dPMP; pyrene 2′-deoxyriboside monophosphate; 5-NI nucleotide; 5-nitro-indolyl-2′-deoxyriboside triphosphate; 5-Nap nucleotide; 5-napthyl-indolyl-2′deoxyriboside triphosphate; 5-Ph nucleotide; 5-phenyl-indolyl-2′-deoxyriboside triphosphate; 5-CE-nucleotide; 5-cyclohexene-indolyl-2′deoxyriboside triphosphate; 5-CH-nucleotide; 5-cyclohexyl-indolyl-2′deoxyriboside triphosphate; dQ; 9-methyl-1; H; -imidazo-[4,5-b]pyridine; dZ; 4-methylbenzimidazole nucleoside; d3FB; 3-fluorobenzene 2′deoxyribosideDNA polymerization; Fidelity; Mutagenesis; Non-natural nucleotides; Hydrogen bonding interactions

Processivity factor of DNA polymerase and its expanding role in normal and translesion DNA synthesis by Zhihao Zhuang; Yongxing Ai (pp. 1081-1093).

Clamp protein or clamp, initially identified as the processivity factor of the replicative DNA polymerase, is indispensable for the timely and faithful replication of DNA genome. Clamp encircles duplex DNA and physically interacts with DNA polymerase. Clamps from different organisms share remarkable similarities in both structure and function. Loading of clamp onto DNA requires the activity of clamp loader. Although all clamp loaders act by converting the chemical energy derived from ATP hydrolysis to mechanical force, intriguing differences exist in the mechanistic details of clamp loading. The structure and function of clamp in normal and translesion DNA synthesis has been subjected to extensive investigations. This review summarizes the current understanding of clamps from three kingdoms of life and the mechanism of loading by their cognate clamp loaders. We also discuss the recent findings on the interactions between clamp and DNA, as well as between clamp and DNA polymerase (both the replicative and specialized DNA polymerases). Lastly the role of clamp in modulating polymerase exchange is discussed in the context of translesion DNA synthesis.

Keywords: DNA polymerase; Clamp loader; PCNA; Polymerase exchange; Translesion DNA synthesis

Single-molecule studies of DNA replisome function by Senthil K. Perumal; Hongjun Yue; Zhenxin Hu; Michelle M. Spiering; Stephen J. Benkovic (pp. 1094-1112).

Fast and accurate replication of DNA is accomplished by the interactions of multiple proteins in the dynamic DNA replisome. The DNA replisome effectively coordinates the leading and lagging strand synthesis of DNA. These complex, yet elegantly organized, molecular machines have been studied extensively by kinetic and structural methods to provide an in-depth understanding of the mechanism of DNA replication. Owing to averaging of observables, unique dynamic information of the biochemical pathways and reactions is concealed in conventional ensemble methods. However, recent advances in the rapidly expanding field of single-molecule analyses to study single biomolecules offer opportunities to probe and understand the dynamic processes involved in large biomolecular complexes such as replisomes. This review will focus on the recent developments in the biochemistry and biophysics of DNA replication employing single-molecule techniques and the insights provided by these methods towards a better understanding of the intricate mechanisms of DNA replication.

Keywords: DNA replication; Polymerase; Replisome; Single-molecule kinetics

Variations on a theme: Eukaryotic Y-family DNA polymerases by M. Todd Washington ⁎; Karissa D. Carlson; Bret D. Freudenthal; John M. Pryor (pp. 1113-1123).

Most classical DNA polymerases, which function in normal DNA replication and repair, are unable to synthesize DNA opposite damage in the template strand. Thus in order to replicate through sites of DNA damage, cells are equipped with a variety of nonclassical DNA polymerases. These nonclassical polymerases differ from their classical counterparts in at least two important respects. First, nonclassical polymerases are able to efficiently incorporate nucleotides opposite DNA lesions while classical polymerases are generally not. Second, nonclassical polymerases synthesize DNA with a substantially lower fidelity than do classical polymerases. Many nonclassical polymerases are members of the Y-family of DNA polymerases, and this article focuses on the mechanisms of the four eukaryotic members of this family: polymerase eta, polymerase kappa, polymerase iota, and the Rev1 protein. We discuss the mechanisms of these enzymes at the kinetic and structural levels with a particular emphasis on how they accommodate damaged DNA substrates. Work over the last decade has shown that the mechanisms of these nonclassical polymerases are fascinating variations of the mechanism of the classical polymerases. The mechanisms of polymerases eta and kappa represent rather minor variations, while the mechanisms of polymerase iota and the Rev1 protein represent rather major variations. These minor and major variations all accomplish the same goal: they allow the nonclassical polymerases to circumvent the problems posed by the template DNA lesion.

Keywords: Abbreviations; 8-oxo-G; 8-oxo-7,8-dihydro-2′-deoxyguanosine; A; 2′-deoxyadenosine; C; 2′-deoxycytidine; dATP; 2′-deoxyadenosine triphosphate; dCTP; 2′-deoxycytidine triphosphate; dGTP; 2′-deoxyguanosine triphosphate; dNTP; 2′deoxynucleoside triphosphate; dTTP; 2′-deoxythymidine triphosphate; G; 2′-deoxyguanosine; PAD; polymerase associated domain; pol; polymerase; PP; _i; pyrophosphate; T; 2′-deoxythymidineTranslesion synthesis; DNA replication; DNA repair; Mutagenesis; Enzyme kinetics; Protein-DNA interactions

Structural diversity of the Y-family DNA polymerases by Janice D. Pata (pp. 1124-1135).

The Y-family translesion DNA polymerases enable cells to tolerate many forms of DNA damage, yet these enzymes have the potential to create genetic mutations at high rates. Although this polymerase family was defined less than a decade ago, more than 90 structures have already been determined so far. These structures show that the individual family members bypass damage and replicate DNA with either error-free or mutagenic outcomes, depending on the polymerase, the lesion and the sequence context. Here, these structures are reviewed and implications for polymerase function are discussed.

Keywords: Error-prone DNA polymerase; Translesion synthesis; Lesion bypass; Mutagenic replication

DNA polymerase Family X: Function, structure, and cellular roles by Jennifer Yamtich; Joann B. Sweasy ⁎ (pp. 1136-1150).

The X family of DNA polymerases in eukaryotic cells consists of terminal transferase and DNA polymerases β, λ, and μ. These enzymes have similar structural portraits, yet different biochemical properties, especially in their interactions with DNA. None of these enzymes possesses a proofreading subdomain, and their intrinsic fidelity of DNA synthesis is much lower than that of a polymerase that functions in cellular DNA replication. In this review, we discuss the similarities and differences of three members of Family X: polymerases β, λ, and μ. We focus on biochemical mechanisms, structural variation, fidelity and lesion bypass mechanisms, and cellular roles. Remarkably, although these enzymes have similar three-dimensional structures, their biochemical properties and cellular functions differ in important ways that impact cellular function.

Keywords: DNA polymerase; Family X; DNA repair; Fidelity of DNA synthesis; Genomic stability; Mutagenesis

Terminal deoxynucleotidyl transferase: The story of a misguided DNA polymerase by Edward A. Motea; Anthony J. Berdis ⁎ (pp. 1151-1166).

Nearly every DNA polymerase characterized to date exclusively catalyzes the incorporation of mononucleotides into a growing primer using a DNA or RNA template as a guide to direct each incorporation event. There is, however, one unique DNA polymerase designated terminal deoxynucleotidyl transferase that performs DNA synthesis using only single-stranded DNA as the nucleic acid substrate. In this chapter, we review the biological role of this enigmatic DNA polymerase and the biochemical mechanism for its ability to perform DNA synthesis in the absence of a templating strand. We compare and contrast the molecular events for template-independent DNA synthesis catalyzed by terminal deoxynucleotidyl transferase with other well-characterized DNA polymerases that perform template-dependent synthesis. This includes a quantitative inspection of how terminal deoxynucleotidyl transferase binds DNA and dNTP substrates, the possible involvement of a conformational change that precedes phosphoryl transfer, and kinetic steps that are associated with the release of products. These enzymatic steps are discussed within the context of the available structures of terminal deoxynucleotidyl transferase in the presence of DNA or nucleotide substrate. In addition, we discuss the ability of proteins involved in replication and recombination to regulate the activity of the terminal deoxynucleotidyl transferase. Finally, the biomedical role of this specialized DNA polymerase is discussed focusing on its involvement in cancer development and its use in biomedical applications such as labeling DNA for detecting apoptosis.

Keywords: Abbreviations; TdT; terminal deoxynucleotidyl transferase; CpG; cytidine guanine base pair; RAG-1; recombination-activating gene 1; RAG-2; recombination-activating gene 2; RSS; recombination signal sequences; Ig; immunoglobulin; NHEJ; non-homologous end-joining; TCR; T cell receptor; PK; protein kinase; PP; _i; inorganic pyrophosphate; 5-NIMP; 5-nitro-indolyl-2′-deoxyriboside-5′-monophosphate; 5-AIMP; 5-amino-indolyl-2′-deoxyriboside-5′-monophosphate; 5-PhIMP; 5-phenyl-indolyl-2′-deoxyriboside-5′-monophosphate; 5-CEIMP; 5-cyclohexenyl-indolyl-2′-deoxyriboside-5′-monophosphate; TdiFs; TdTase interacting factors; PCNA; proliferating cell nuclear antigenDNA polymerization; Template-independent DNA synthesis; Fidelity; Recombination; Nucleotide analog

Coordinating DNA polymerase traffic during high and low fidelity synthesis by Mark D. Sutton (pp. 1167-1179).

With the discovery that organisms possess multiple DNA polymerases (Pols) displaying different fidelities, processivities, and activities came the realization that mechanisms must exist to manage the actions of these diverse enzymes to prevent gratuitous mutations. Although many of the Pols encoded by most organisms are largely accurate, and participate in DNA replication and DNA repair, a sizeable fraction display a reduced fidelity, and act to catalyze potentially error-prone translesion DNA synthesis (TLS) past lesions that persist in the DNA. Striking the proper balance between use of these different enzymes during DNA replication, DNA repair, and TLS is essential for ensuring accurate duplication of the cell's genome. This review highlights mechanisms that organisms utilize to manage the actions of their different Pols. A particular emphasis is placed on discussion of current models for how different Pols switch places with each other at the replication fork during high fidelity replication and potentially error-pone TLS.

Keywords: Sliding clamp; DNA polymerase; DNA replication; Translesion DNA synthesis; Mutagenesis; Polymerase switch; Toolbelt

Mechanism and evolution of DNA primases by Robert D. Kuchta ⁎; Gudrun Stengel (pp. 1180-1189).

DNA primase synthesizes short RNA primers that replicative polymerases further elongate in order to initiate the synthesis of all new DNA strands. Thus, primase owes its existence to the inability of DNA polymerases to initiate DNA synthesis starting with 2 dNTPs. Here, we discuss the evolutionary relationships between the different families of primases (viral, eubacterial, archael, and eukaryotic) and the catalytic mechanisms of these enzymes. This includes how they choose an initiation site, elongate the growing primer, and then only synthesize primers of defined length via an inherent ability to count. Finally, the low fidelity of primases along with the development of primase inhibitors is described.

Keywords: Primase; Polymerase; NTP; dNTP; Mechanism; Inhibition; Counting; Fidelity; Misincorporation; Initiation

Structures of telomerase subunits provide functional insights by Vijay G. Sekaran; Joana Soares; Michael B. Jarstfer (pp. 1190-1201).

Background: Telomerase continues to generate substantial attention both because of its pivotal roles in cellular proliferation and aging and because of its unusual structure and mechanism. By replenishing telomeric DNA lost during the cell cycle, telomerase overcomes one of the many hurdles facing cellular immortalization. Functionally, telomerase is a reverse transcriptase, and it shares structural and mechanistic features with this class of nucleotide polymerases. Telomerase is a very unusual reverse transcriptase because it remains stably associated with its template and because it reverse transcribes multiple copies of its template onto a single primer in one reaction cycle. Scope of review: Here, we review recent findings that illuminate our understanding of telomerase. Even though the specific emphasis is on structure and mechanism, we also highlight new insights into the roles of telomerase in human biology. General significance: Recent advances in the structural biology of telomerase, including high resolution structures of the catalytic subunit of a beetle telomerase and two domains of a ciliate telomerase catalytic subunit, provide new perspectives into telomerase biochemistry and reveal new puzzles.

Keywords: Abbreviations; TERT; Telomerase Reverse Transcriptase; TER; Telomerase RNA; dNTPs; Deoxyribonucleotide triphosphates; TEN; Telomerase Essential N-terminal; POT1; Protection of Telomeres 1, TPP1, PTOP, PIP1, or TINT1; TCAB1; Telomerase Cajal Body 1, RID1, RNA-Interaction Domain 1; RID2; RNA-Interaction Domain 2; TRBD; TER-binding domain; IFD; Insertion in Fingers DomainTelomerase; Telomere; Reverse transcriptase

Reverse transcriptase in motion: Conformational dynamics of enzyme–substrate interactions by Gotte Matthias Götte; Jason W. Rausch; Bruno Marchand; Stefan Sarafianos; Stuart F.J. Le Grice (pp. 1202-1212).

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) catalyzes synthesis of integration-competent, double-stranded DNA from the single-stranded viral RNA genome, combining both polymerizing and hydrolytic functions to synthesize approximately 20,000 phosphodiester bonds. Despite a wealth of biochemical studies, the manner whereby the enzyme adopts different orientations to coordinate its DNA polymerase and ribonuclease (RNase) H activities has remained elusive. Likewise, the lower processivity of HIV-1 RT raises the issue of polymerization site targeting, should the enzyme re-engage its nucleic acid substrate several hundred nucleotides from the primer terminus. Although X-ray crystallography has clearly contributed to our understanding of RT-containing nucleoprotein complexes, it provides a static picture, revealing few details regarding motion of the enzyme on the substrate. Recent development of site-specific footprinting and the application of single molecule spectroscopy have allowed us to follow individual steps in the reverse transcription process with significantly greater precision. Progress in these areas and the implications for investigational and established inhibitors that interfere with RT motion on nucleic acid is reviewed here.

Keywords: Retrovirus; Reverse transcriptase; Ribonuclease H; Translocational equilibrium; Single molecule spectroscopy

A mechanistic view of human mitochondrial DNA polymerase γ: Providing insight into drug toxicity and mitochondrial disease by Christopher M. Bailey; Karen S. Anderson (pp. 1213-1222).

Mitochondrial DNA polymerase gamma (Pol γ) is the sole polymerase responsible for replication of the mitochondrial genome. The study of human Pol γ is of key importance to clinically relevant issues such as nucleoside analog toxicity and mitochondrial disorders such as progressive external ophthalmoplegia. The development of a recombinant form of the human Pol γ holoenzyme provided an essential tool in understanding the mechanism of these clinically relevant phenomena using kinetic methodologies. This review will provide a brief history on the discovery and characterization of human mitochondrial DNA polymerase γ, focusing on kinetic analyses of the polymerase and mechanistic data illustrating structure–function relationships to explain drug toxicity and mitochondrial disease.

Keywords: DNA polymerase gamma; Mitochondrial genome; Nucleoside analog toxicity; Progressive external ophthalmoplegia; Pre-steady-state kinetics

Modifications to the dNTP triphosphate moiety: From mechanistic probes for DNA polymerases to antiviral and anti-cancer drug design by Charles E. McKenna; Boris A. Kashemirov; Larryn W. Peterson; Myron F. Goodman (pp. 1223-1230).

Abnormal replication of DNA is associated with many important human diseases, most notably viral infections and neoplasms. Existing approaches to chemotherapeutics for diseases associated with dysfunctional DNA replication classically involve nucleoside analogues that inhibit polymerase activity due to modification in the nucleobase and/or ribose moieties. These compounds must undergo multiple phosphorylation steps in vivo, converting them into triphosphosphates, in order to inhibit their targeted DNA polymerase. Nucleotide monophosphonates enable bypassing the initial phosphorylation step at the cost of decreased bioavailability. Relatively little attention has been paid to higher nucleotides (corresponding to the natural di- and triphosphate DNA polymerase substrates) as drug platforms due to their expected poor deliverability. However, a better understanding of DNA polymerase mechanism and fidelity dependence on the triphosphate moiety is beginning to emerge, aided by systematic incorporation into this group of substituted methylenebisphosphonate probes. Meanwhile, other bridging, as well as non-bridging, modifications have revealed intriguing possibilities for new drug design. We briefly survey some of this recent work, and argue that the potential of nucleotide-based drugs, and intriguing preliminary progress in this area, warrant acceptance of the challenges that they present with respect to bioavailability and metabolic stability.

Keywords: Abbreviations; ANS; 1-aminonaphthalene-5-sulfonate; AZTMP; AZT-monophosphate; AZTDP; AZT-diphosphate; AZTTP; AZT-triphosphate; BER; base excision repair; DNA; deoxynucleic acid; dNTP; deoxynucleoside triphosphate; G; guanosine; HAART; highly active antiretroviral therapy; HAP; hydroxyapatite; HIV; human immunodeficiency virus; LFER; linear free energy relationship; N-BPs; nitrogen-containing bisphosphonates; NRTI; nucleoside/nucleotide reverse transcriptase inhibitor; NNRTI; non-nucleoside/nucleotide reverse transcriptase inhibitor; PI; protease inhibitors; T; thymidine; TMPK; human thymidylate kinase; NATP; nucleoside analogue triphosphate; pol β; DNA polymerase β; pol γ; DNA polymerase γ; pol δ; DNA polymerase δ; pol ε; DNA polymerase ε; pol η; DNA polymerase η; pol ι; DNA polymerase ι; VEGF; vascular endothelial growth factorDNA polymerase; Nucleotide analogue; Bisphosphonate; Antiviral; Anti-cancer; Drug targeting; Drug delivery

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BBA - Proteins and Proteomics (v.1804, #5)