Enzymology
Polymerase γ efficiently replicates through many natural template barriers but stalls at the HSP1 quadruplex
Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters dsDNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the polymerase γ heterotrimer is well-adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks.Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis
Magnesium ions play a critical role in catalysis by many enzymes and contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg2+ ions in each step in the catalysis have not been resolved. We first examined the roles of Mg2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase. We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg2+ concentration.Kinetic investigation of the polymerase and exonuclease activities of human DNA polymerase ε holoenzyme
In eukaryotic DNA replication, DNA polymerase ε (Polε) is responsible for leading strand synthesis, whereas DNA polymerases α and δ synthesize the lagging strand. The human Polε (hPolε) holoenzyme is comprised of the catalytic p261 subunit and the noncatalytic p59, p17, and p12 small subunits. So far, the contribution of the noncatalytic subunits to hPolε function is not well understood. Using pre-steady-state kinetic methods, we established a minimal kinetic mechanism for DNA polymerization and editing catalyzed by the hPolε holoenzyme.Conformational dynamics during high-fidelity DNA replication and translocation defined using a DNA polymerase with a fluorescent artificial amino acid
We address the role of enzyme conformational dynamics in specificity for a high-fidelity DNA polymerase responsible for genome replication. We present the complete characterization of the conformational dynamics during the correct nucleotide incorporation forward and reverse reactions using stopped-flow and rapid-quench methods with a T7 DNA polymerase variant containing a fluorescent unnatural amino acid, (7-hydroxy-4-coumarin-yl) ethylglycine, which provides a signal for enzyme conformational changes.Pif1, RPA, and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis
Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ.DnaB helicase is recruited to the replication initiation complex via binding of DnaA domain I to the lateral surface of the DnaB N-terminal domain
The DNA replication protein DnaA in Escherichia coli constructs higher-order complexes on the origin, oriC, to unwind this region. DnaB helicase is loaded onto unwound oriC via interactions with the DnaC loader and the DnaA complex. The DnaB–DnaC complex is recruited to the DnaA complex via stable binding of DnaB to DnaA domain I. The DnaB–DnaC complex is then directed to unwound oriC via a weak interaction between DnaB and DnaA domain III. Previously, we showed that Phe46 in DnaA domain I binds to DnaB.The many lives of type IA topoisomerases
The double-helical structure of genomic DNA is both elegant and functional in that it serves both to protect vulnerable DNA bases and to facilitate DNA replication and compaction. However, these design advantages come at the cost of having to evolve and maintain a cellular machinery that can manipulate a long polymeric molecule that readily becomes topologically entangled whenever it has to be opened for translation, replication, or repair. If such a machinery fails to eliminate detrimental topological entanglements, utilization of the information stored in the DNA double helix is compromised.Human replication protein A induces dynamic changes in single-stranded DNA and RNA structures
Replication protein A (RPA) is the major eukaryotic ssDNA-binding protein and has essential roles in genome maintenance. RPA binds to ssDNA through multiple modes, and recent studies have suggested that the RPA–ssDNA interaction is dynamic. However, how RPA alternates between different binding modes and modifies ssDNA structures in this dynamic interaction remains unknown. Here, we used single-molecule FRET to systematically investigate the interaction between human RPA and ssDNA. We show that RPA can adopt different types of binding complexes with ssDNAs of different lengths, leading to the straightening or bending of the ssDNAs, depending on both the length and structure of the ssDNA substrate and the RPA concentration.Glutathione-glutaredoxin is an efficient electron donor system for mammalian p53R2–R1-dependent ribonucleotide reductase
Deoxyribonucleotides are DNA building blocks and are produced de novo by reduction of ribose to deoxyribose. This reduction is catalyzed by ribonucleotide reductase (RNR), a heterodimeric tetramer enzyme in mammalian cells, having one of two free radical-containing subunits called R2 and p53R2. R2 is S-phase specific and used for DNA replication, whereas p53R2 functions in DNA repair and mitochondrial DNA synthesis. The larger RNR subunit, R1, has catalytically active cysteine thiols in its buried active site and a C-terminal swinging arm, with a Cys-Leu-Met-Cys sequence suggested to act as a shuttle dithiol/disulfide for electron transport.The abundant DNA adduct N7-methyl deoxyguanosine contributes to miscoding during replication by human DNA polymerase η
Aside from abasic sites and ribonucleotides, the DNA adduct N7-methyl deoxyguanosine (N7-CH3 dG) is one of the most abundant lesions in mammalian DNA. Because N7-CH3 dG is unstable, leading to deglycosylation and ring-opening, its miscoding potential is not well-understood. Here, we employed a 2′-fluoro isostere approach to synthesize an oligonucleotide containing an analog of this lesion (N7-CH3 2′-F dG) and examined its miscoding potential with four Y-family translesion synthesis DNA polymerases (pols): human pol (hpol) η, hpol κ, and hpol ι and Dpo4 from the archaeal thermophile Sulfolobus solfataricus.Human DNA polymerase η has reverse transcriptase activity in cellular environments
Classical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol η (hpol η) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol η residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity.Replication protein A dynamically regulates monoubiquitination of proliferating cell nuclear antigen
DNA damage tolerance permits bypass of DNA lesions encountered during S-phase and may be carried out by translesion DNA synthesis (TLS). Human TLS requires selective monoubiquitination of proliferating cell nuclear antigen (PCNA) sliding clamps encircling damaged DNA. This posttranslational modification (PTM) is catalyzed by Rad6/Rad18. Recent studies revealed that replication protein A (RPA), the major ssDNA-binding protein, is involved in the regulation of PCNA monoubiquitination and interacts directly with Rad18 on chromatin and in the nucleoplasm.The HIRAN domain of helicase-like transcription factor positions the DNA translocase motor to drive efficient DNA fork regression
Helicase-like transcription factor (HLTF) is a central mediator of the DNA damage response and maintains genome stability by regressing stalled replication forks. The N-terminal HIRAN domain binds specifically to the 3′-end of single-stranded DNA (ssDNA), and disrupting this function interferes with fork regression in vitro as well as replication fork progression in cells under replication stress. Here, we investigated the mechanism by which the HIRAN-ssDNA interaction facilitates fork remodeling.Polymerase θ-helicase efficiently unwinds DNA and RNA-DNA hybrids
POLQ is a unique multifunctional replication and repair gene that encodes for a N-terminal superfamily 2 helicase and a C-terminal A-family polymerase. Although the function of the polymerase domain has been investigated, little is understood regarding the helicase domain. Multiple studies have reported that polymerase θ-helicase (Polθ-helicase) is unable to unwind DNA. However, it exhibits ATPase activity that is stimulated by single-stranded DNA, which presents a biochemical conundrum. In contrast to previous reports, we demonstrate that Polθ-helicase (residues 1–894) efficiently unwinds DNA with 3′–5′ polarity, including DNA with 3′ or 5′ overhangs, blunt-ended DNA, and replication forks.Amino acid residues in HIV-2 reverse transcriptase that restrict the development of nucleoside analogue resistance through the excision pathway
Nucleoside reverse transcriptase (RT) inhibitors (NRTIs) are the backbone of current antiretroviral treatments. However, the emergence of viral resistance against NRTIs is a major threat to their therapeutic effectiveness. In HIV-1, NRTI resistance-associated mutations either reduce RT-mediated incorporation of NRTI triphosphates (discrimination mechanism) or confer an ATP-mediated nucleotide excision activity that removes the inhibitor from the 3′ terminus of DNA primers, enabling further primer elongation (excision mechanism).Positioning the 5′-flap junction in the active site controls the rate of flap endonuclease-1–catalyzed DNA cleavage
Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair, and recombination and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5′-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise.Linchpin DNA-binding residues serve as go/no-go controls in the replication factor C-catalyzed clamp-loading mechanism
DNA polymerases depend on circular sliding clamps for processive replication. Clamps must be loaded onto primer–template DNA (ptDNA) by clamp loaders that open and close clamps around ptDNA in an ATP-fueled reaction. All clamp loaders share a core structure in which five subunits form a spiral chamber that binds the clamp at its base in a twisted open form and encloses ptDNA within, while binding and hydrolyzing ATP to topologically link the clamp and ptDNA. To understand how clamp loaders perform this complex task, here we focused on conserved arginines that might play a central coordinating role in the mechanism because they can alternately contact ptDNA or Walker B glutamate in the ATPase site and lie close to the clamp loader–clamp-binding interface.Kinetic analyses of single-stranded break repair by human DNA ligase III isoforms reveal biochemical differences from DNA ligase I
Humans have three genes encoding DNA ligases with conserved structural features and activities, but they also have notable differences. The LIG3 gene encodes a ubiquitous isoform in all tissues (LIG3α) and a germ line–specific splicing isoform (LIG3β) that differs in the C-terminal domain. Both isoforms are found in the nucleus and the mitochondria. Here, we determined the kinetics and thermodynamics of single-stranded break ligation by LIG3α and LIG3β and compared this framework to that of LIG1, the nuclear replicative ligase.Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea
Incorporation of ribonucleotides during DNA replication has severe consequences for genome stability. Although eukaryotes possess a number of redundancies for initiating and completing repair of misincorporated ribonucleotides, archaea such as Thermococcus rely only upon RNaseH2 to initiate the pathway. Because Thermococcus DNA polymerases incorporate as many as 1,000 ribonucleotides per genome, RNaseH2 must be efficient at recognizing and nicking at embedded ribonucleotides to ensure genome integrity.Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells
During DNA replication in eukaryotic cells, short single-stranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. The Okazaki fragments originate from ∼35-nucleotide-long RNA-DNA primers. After Okazaki fragment synthesis, these primers must be removed to allow fragment joining into a continuous lagging strand. To date, the models of enzymatic machinery that removes the RNA-DNA primers have come almost exclusively from biochemical reconstitution studies and some genetic interaction assays, and there is little direct evidence to confirm these models.Residues in the RecQ C-terminal Domain of the Human Werner Syndrome Helicase Are Involved in Unwinding G-quadruplex DNA
The structural and biophysical properties typically associated with G-quadruplex (G4) structures render them a significant block for DNA replication, which must be overcome for cell division to occur. The Werner syndrome protein (WRN) is a RecQ family helicase that has been implicated in the efficient processing of G4 DNA structures. The aim of this study was to identify the residues of WRN involved in the binding and ATPase-driven unwinding of G4 DNA. Using a c-Myc G4 DNA model sequence and recombinant WRN, we have determined that the RecQ-C-terminal (RQC) domain of WRN imparts a 2-fold preference for binding to G4 DNA relative to non-G4 DNA substrates.Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks
Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5′ end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5′-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks.Cooperative DnaA Binding to the Negatively Supercoiled datA Locus Stimulates DnaA-ATP Hydrolysis
Timely initiation of replication in Escherichia coli requires functional regulation of the replication initiator, ATP-DnaA. The cellular level of ATP-DnaA increases just before initiation, after which its level decreases through hydrolysis of DnaA-bound ATP, yielding initiation-inactive ADP-DnaA. Previously, we reported a novel DnaA-ATP hydrolysis system involving the chromosomal locus datA and named it datA-dependent DnaA-ATP hydrolysis (DDAH). The datA locus contains a binding site for a nucleoid-associating factor integration host factor (IHF) and a cluster of three known DnaA-binding sites, which are important for DDAH.Rate-limiting Pyrophosphate Release by HIV Reverse Transcriptase Improves Fidelity
Previous measurements of the rates of polymerization and pyrophosphate release with DNA templates showed that pyrophosphate (PPi) dissociation was fast after nucleotide incorporation so that it did not contribute to enzyme specificity (kcat/Km). Here, kinetic parameters governing nucleotide incorporation and PPi release were determined using an RNA template. Compared with a DNA template of the same sequence, the rate of chemistry increased by up to 10-fold (250 versus 24 s−1), whereas the rate of PPi release decreased to approximately 58 s−1 so that PPi release became the rate-limiting step.The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA
Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1.
