Showing papers on "DNA clamp published in 2004"
TL;DR: This work shows that, in human cells, PCNA becomes monoubiquitinated following UV irradiation of the cells and that this is dependent on the hRad18 protein, and identifies two motifs in poleta that are involved in this interaction.
870 citations
TL;DR: It is documented that single-stranded DNA adsorbs on negatively charged gold nanoparticles (Au-nps) with a rate that depends on sequence length and temperature, which forms the basis for a colorimetric assay to identify specific sequences and single nucleotide polymorphisms on polymerase chain reaction (PCR)-amplified DNA.
Abstract: We document the surprising result that single-stranded DNA adsorbs on negatively charged gold nanoparticles (Au-nps) with a rate that depends on sequence length and temperature. After ss-DNA adsorbs on Au-nps, we find that the particles are stabilized against salt-induced aggregation. These observations can be rationalized on the basis of electrostatics and form the basis for a colorimetric assay to identify specific sequences and single nucleotide polymorphisms on polymerase chain reaction (PCR)-amplified DNA. The assay is label-free, requires no covalent modification of the DNA or Au-np surfaces, and takes on the sensitivity of PCR. Most important, binding of target and probe takes place in solution where hybridization occurs in less than 1 min. As an example, we test PCR-amplified genomic DNA from clinical samples for single nucleotide polymorphisms (SNPs) associated with a fatal arrhythmia known as long QT syndrome.
627 citations
TL;DR: It is demonstrated that unmethylated sites embedded in a hemimethylated context are modified at an approximately 24-fold reduced rate, which demonstrates that the enzyme accurately copies existing patterns of methylation.
514 citations
TL;DR: It is shown that intracellular transcription of G-rich regions produces novel DNA structures, visible by electron microscopy as large G-loops formed cotranscriptionally, and they contain G4 DNA on one strand and a stable RNA/DNA hybrid on the other.
Abstract: We show that intracellular transcription of G-rich regions produces novel DNA structures, visible by electron microscopy as large (150–500 bp) loops. These G-loops are formed cotranscriptionally, and they contain G4 DNA on one strand and a stable RNA/DNA hybrid on the other. G-loop formation requires a G-rich nontemplate strand and reflects the unusual stability of the rG/dC base pair. G-loops and G4 DNA form efficiently within plasmid genomes transcribed in vitro or in Escherichia coli. These results establish that G4 DNA can form in vivo, a finding with implications for stability and maintenance of all G-rich genomic regions.
468 citations
TL;DR: This model, in which the clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer–template junctions by the RFC results in ATP hydrolysis and release of the sliding clamp on DNA.
Abstract: Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. Here we report the crystal structure of the five-protein clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding clamp (proliferating cell nuclear antigen, PCNA). Tight interfacial coordination of the ATP analogue ATP-gammaS by RFC results in a spiral arrangement of the ATPase domains of the clamp loader above the PCNA ring. Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA double helix. This model, in which the clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer-template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding clamp on DNA.
439 citations
TL;DR: The results support the attractive possibility that hMSH4-hMSH5 stabilizes and preserves a meiotic bimolecular double-strand break repair (DSBR) intermediate.
385 citations
TL;DR: The two-subunit holoenzyme is an efficient and processive polymerase, which exhibits high fidelity in nucleotide selection and incorporation while proofreading errors with its intrinsic 3′ → 5′ exonuclease.
Abstract: DNA polymerase (pol) gamma is the sole DNA polymerase in animal mitochondria. Biochemical and genetic evidence document a key role for pol gamma in mitochondrial DNA replication, and whereas DNA repair and recombination were thought to be limited or absent in animal mitochondria, both have been demonstrated in recent years. Thus, the mitochondrial replicase is also apparently responsible for the relevant DNA synthetic reactions in these processes. Pol gamma comprises a catalytic core in a heterodimeric complex with an accessory subunit. The two-subunit holoenzyme is an efficient and processive polymerase, which exhibits high fidelity in nucleotide selection and incorporation while proofreading errors with its intrinsic 3' 5' exonuclease. Incorporation of nucleotide analogs followed by proofreading failure leads to mitochondrial toxicity in antiviral therapy, and misincorporation during DNA replication leads to mitochondrial mutagenesis and dysfunction. This review describes our current understanding of pol gamma biochemistry and biology, and it introduces other key proteins that function at the mitochondrial DNA replication fork.
377 citations
TL;DR: These findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork, and explain why mutations in these two proteins cause an identical syndrome.
Abstract: We here reconstitute a minimal mammalian mitochondrial DNA (mtDNA) replisome in vitro. The mtDNA polymerase (POLγ) cannot use double-stranded DNA (dsDNA) as template for DNA synthesis. Similarly, the TWINKLE DNA helicase is unable to unwind longer stretches of dsDNA. In combination, POLγ and TWINKLE form a processive replication machinery, which can use dsDNA as template to synthesize single-stranded DNA (ssDNA) molecules of about 2 kb. The addition of the mitochondrial ssDNA-binding protein stimulates the reaction further, generating DNA products of about 16 kb, the size of the mammalian mtDNA molecule. The observed DNA synthesis rate is 180 base pairs (bp)/min, corresponding closely to the previously calculated value of 270 bp/min for in vivo DNA replication. Our findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork. Furthermore, mutations in TWINKLE and POLγ cause autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with deletions in mitochondrial DNA. The functional interactions between TWINKLE and POLγ thus explain why mutations in these two proteins cause an identical syndrome.
375 citations
TL;DR: Findings suggest that the 9-1-1 clamp is a multifunctional complex that is loaded onto DNA at sites of damage, where it coordinates checkpoint activation and DNA repair.
339 citations
TL;DR: The crystal structure of human DNA ligase I in complex with a nicked, 5′ adenylated DNA intermediate shows that the enzyme redirects the path of the double helix to expose the nick termini for the strand-joining reaction and reveals a unique feature of mammalian ligases: a DNA-binding domain that allows ligases I to encircle its DNA substrate, stabilizes the DNA in a distorted structure, and positions the catalytic core on the nick.
Abstract: The end-joining reaction catalysed by DNA ligases is required by all organisms and serves as the ultimate step of DNA replication, repair and recombination processes. One of three well characterized mammalian DNA ligases, DNA ligase I, joins Okazaki fragments during DNA replication. Here we report the crystal structure of human DNA ligase I (residues 233 to 919) in complex with a nicked, 5' adenylated DNA intermediate. The structure shows that the enzyme redirects the path of the double helix to expose the nick termini for the strand-joining reaction. It also reveals a unique feature of mammalian ligases: a DNA-binding domain that allows ligase I to encircle its DNA substrate, stabilizes the DNA in a distorted structure, and positions the catalytic core on the nick. Similarities in the toroidal shape and dimensions of DNA ligase I and the proliferating cell nuclear antigen sliding clamp are suggestive of an extensive protein-protein interface that may coordinate the joining of Okazaki fragments.
321 citations
TL;DR: Multiple stepwise ATP-binding events to RFC are required to load PCNA onto primed DNA, and this stepwise mechanism should permit editing of this process at individual steps and allow for divergence of the default process into more specialized modes.
Abstract: The proliferating cell nuclear antigen PCNA functions at multiple levels in directing DNA metabolic pathways. Unbound to DNA, PCNA promotes localization of replication factors with a consensus PCNA-binding domain to replication factories. When bound to DNA, PCNA organizes various proteins involved in DNA replication, DNA repair, DNA modification, and chromatin modeling. Its modification by ubiquitin directs the cellular response to DNA damage. The ring-like PCNA homotrimer encircles double-stranded DNA and slides spontaneously across it. Loading of PCNA onto DNA at template-primer junctions is performed in an ATP-dependent process by replication factor C (RFC), a heteropentameric AAA+ protein complex consisting of the Rfc1, Rfc2, Rfc3, Rfc4, and Rfc5 subunits. Loading of yeast PCNA (POL30) is mechanistically distinct from analogous processes in E. coli (beta subunit by the gamma complex) and bacteriophage T4 (gp45 by gp44/62). Multiple stepwise ATP-binding events to RFC are required to load PCNA onto primed DNA. This stepwise mechanism should permit editing of this process at individual steps and allow for divergence of the default process into more specialized modes. Indeed, alternative RFC complexes consisting of the small RFC subunits together with an alternative Rfc1-like subunit have been identified. A complex required for the DNA damage checkpoint contains the Rad24 subunit, a complex required for sister chromatid cohesion contains the Ctf18 subunit, and a complex that aids in genome stability contains the Elg1 subunit. Only the RFC-Rad24 complex has a known associated clamp, a heterotrimeric complex consisting of Rad17, Mec3, and Ddc1. The other putative clamp loaders could either act on clamps yet to be identified or act on the two known clamps.
TL;DR: Comparisons reveal a fundamental mechanism of error-prone replication and show how 8oxoG, and DNA lesions in general, can form mismatches that evade polymerase error-detection mechanisms, potentially leading to the stable incorporation of lethal mutations.
Abstract: Aerobic respiration generates reactive oxygen species that can damage guanine residues and lead to the production of 8-oxoguanine (8oxoG), the major mutagenic oxidative lesion in the genome. Oxidative damage is implicated in ageing and cancer, and its prevalence presents a constant challenge to DNA polymerases that ensure accurate transmission of genomic information. When these polymerases encounter 8oxoG, they frequently catalyse misincorporation of adenine in preference to accurate incorporation of cytosine. This results in the propagation of G to T transversions, which are commonly observed somatic mutations associated with human cancers. Here, we present sequential snapshots of a high-fidelity DNA polymerase during both accurate and mutagenic replication of 8oxoG. Comparison of these crystal structures reveals that 8oxoG induces an inversion of the mismatch recognition mechanisms that normally proofread DNA, such that the 8oxoG.adenine mismatch mimics a cognate base pair whereas the 8oxoG.cytosine base pair behaves as a mismatch. These studies reveal a fundamental mechanism of error-prone replication and show how 8oxoG, and DNA lesions in general, can form mismatches that evade polymerase error-detection mechanisms, potentially leading to the stable incorporation of lethal mutations.
TL;DR: Current data indicates that hyper-phosphorylation causes a change in RPA conformation that down-regulates activity in DNA replication but does not affect DNA repair processes, which suggests that the role of RPA phosphorylation in the cellular response to DNA damage is to help regulate DNA metabolism and promote DNA repair.
TL;DR: A structural characterization of all 12 possible mismatches captured at the growing primer terminus in the active site of a polymerase is presented, suggesting the structural basis for the short-term memory of replication errors.
TL;DR: The DNA and protein conformational changes, composite complex structures, FRET, and mutational results support enzyme-PCNA alignments and a kinked DNA pivot point that appear suitable to coordinate rotary handoffs of kinksed DNA intermediates among enzymes localized by the three PCNA binding sites.
TL;DR: It is shown that polynucleotide kinase (PNK), with its ability to process ionizing radiation‐induced 5′‐OH and 3′‐phosphate DNA termini, functions in NHEJ via an FHA‐dependent interaction with CK2‐ phosphorylated Xrcc4, and that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains.
Abstract: Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4–DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5′-OH and 3′-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA–Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4–PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.
TL;DR: The results suggest that polymerase λ is the primary gap-filling polymerase for accurate nonhomologous end joining, and that the Brca1 C-terminal domain is required for this activity.
TL;DR: In this article, a yeast two-hybrid screen was used to identify proteins that interact with REV1 and showed that hREV1 interacts with hpolη and hpolκ.
TL;DR: These studies highlight a remarkably efficient mechanism for Okazaki fragment maturation in which Pol delta by default displaces 2-3 nt of any downstream RNA or DNA it encounters.
Abstract: During each yeast cell cycle, ∼100,000 nicks are generated during lagging-strand DNA replication. Efficient nick processing during Okazaki fragment maturation requires the coordinated action of DNA polymerase (Pol ) and the FLAP endonuclease FEN1. Misregulation of this process leads to the accumulation of double-stranded breaks and cell lethality. Our studies highlight a remarkably efficient mechanism for Okazaki fragment maturation in which Pol by default displaces 2–3 nt of any downstream RNA or DNA it encounters. In the presence of FEN1, efficient nick translation ensues, whereby a mixture of mono- and small oligonucleotides are released. If FEN1 is absent or not optimally functional, the ability of Pol to back up via its 3–5-exonuclease activity, a process called idling, maintains the polymerase at a position that is ideal either for ligation (in case of a DNA–DNA nick) or for subsequent engagement by FEN1 (in case of a DNA–RNA nick). Consistent with the hypothesis that DNA polymerase is the leading-strand enzyme, we observed no idling by this enzyme and no cooperation with FEN1 for creating a ligatable nick.
TL;DR: The PIP-box and flanking regions provide a small docking peptide whose affinities can be readily adjusted in accord with biological necessity to mediate the binding of DNA replication and repair proteins to hPCNA.
TL;DR: The crystal structure of the polymerase with 8oG templating dC insertion shows that the O8 oxygen is tolerated by strong kinking of the DNA template, explaining why translesion synthesis is permitted without proofreading of an8oG·dA mismatch, thus providing insight into the high mutagenic potential of 8o G.
Abstract: Accurate DNA replication involves polymerases with high nucleotide selectivity and proofreading activity. We show here why both fidelity mechanisms fail when normally accurate T7 DNA polymerase bypasses the common oxidative lesion 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8oG). The crystal structure of the polymerase with 8oG templating dC insertion shows that the O8 oxygen is tolerated by strong kinking of the DNA template. A model of a corresponding structure with dATP predicts steric and electrostatic clashes that would reduce but not eliminate insertion of dA. The structure of a postinsertional complex shows 8oG(syn)·dA (anti) in a Hoogsteen-like base pair at the 3′ terminus, and polymerase interactions with the minor groove surface of the mismatch that mimic those with undamaged, matched base pairs. This explains why translesion synthesis is permitted without proofreading of an 8oG·dA mismatch, thus providing insight into the high mutagenic potential of 8oG.
TL;DR: A single-molecule manipulation technique is used to monitor real-time changes in extension of a single, stretched, nicked dsDNA substrate as it is unwound by a single enzyme, and observes a feature not seen in bulk assays: unwinding is preferentially followed by a slow, enzyme-translocation-limited rezipping of the separated strands.
Abstract: DNA helicases are enzymes capable of unwinding double-stranded DNA (dsDNA) to provide the single-stranded DNA template required in many biological processes. Among these, UvrD, an essential DNA repair enzyme, has been shown to unwind dsDNA while moving 3′-5′ on one strand. Here, we use a single-molecule manipulation technique to monitor real-time changes in extension of a single, stretched, nicked dsDNA substrate as it is unwound by a single enzyme. This technique offers a means for measuring the rate, lifetime, and processivity of the enzymatic complex as a function of ATP, and for estimating the helicase step size. Strikingly, we observe a feature not seen in bulk assays: unwinding is preferentially followed by a slow, enzyme-translocation-limited rezipping of the separated strands rather than by dissociation of the enzymatic complex followed by quick rehybridization of the DNA strands. We address the mechanism underlying this phenomenon and propose a fully characterized model in which UvrD switches strands and translocates backwards on the other strand, allowing the DNA to reanneal in its wake.
TL;DR: The crystal structure of phi29 DNA polymerase determined at 2.2 A resolution provides explanations for its extraordinary processivity and strand displacement activities.
TL;DR: Domain analysis of Pol32, the third subunit of Saccharomyces cerevisiae DNA polymerase δ, shows that during DNA synthesis the carboxyl-terminal domain of Pol 32 interacts with the car boxyl-Terminal region of PCNA, whereas interactions of the other subunit(s) of Pol δ localize largely to a hydrophobic pocket at the interdomain connector loop region ofPCNA.
TL;DR: It is shown that hDmc1 mediates strand exchange between paired DNA substrates over at least several thousand base pairs, which is probably indispensable for repair of DNA double-strand breaks during meiosis and for maintaining the ploidy of meiotic chromosomes.
Abstract: Homologous recombination is crucial for the repair of DNA breaks and maintenance of genome stability. In Escherichia coli, homologous recombination is dependent on the RecA protein. In the presence of ATP, RecA mediates the homologous DNA pairing and strand exchange reaction that links recombining DNA molecules. DNA joint formation is initiated through the nucleation of RecA onto single-stranded DNA (ssDNA) to form helical nucleoprotein filaments. Two RecA-like recombinases, Rad51 and Dmc1, exist in eukaryotes. Whereas Rad51 is needed for both mitotic and meiotic recombination events, the function of Dmc1 is restricted to meiosis. Here we examine human Dmc1 protein (hDmc1) for the ability to promote DNA strand exchange, and show that hDmc1 mediates strand exchange between paired DNA substrates over at least several thousand base pairs. DNA strand exchange requires ATP and is strongly dependent on the heterotrimeric ssDNA-binding molecule replication factor A (RPA). We present evidence that hDmc1-mediated DNA recombination initiates through the nucleation of hDmc1 onto ssDNA to form a helical nucleoprotein filament. The DNA strand exchange activity of hDmc1 is probably indispensable for repair of DNA double-strand breaks during meiosis and for maintaining the ploidy of meiotic chromosomes.
TL;DR: It is found that the rate of formation and the stability of the unwound complex depend profoundly on super coiling and that supercoiling exerts its effects mechanically (through torque), and not structurally (through the number and position of supercoils).
Abstract: By monitoring the end-to-end extension of a mechanically stretched, supercoiled, single DNA molecule, we have been able directly to observe the change in extension associated with unwinding of approximately one turn of promoter DNA by RNA polymerase (RNAP) By performing parallel experiments with negatively and positively supercoiled DNA, we have been able to deconvolute the change in extension caused by RNAP-dependent DNA unwinding (with ≈1-bp resolution) and the change in extension caused by RNAP-dependent DNA compaction (with ≈5-nm resolution) We have used this approach to quantify the extent of unwinding and compaction, the kinetics of unwinding and compaction, and effects of supercoiling, sequence, ppGpp, and nucleotides We also have used this approach to detect promoter clearance and promoter recycling by successive RNAP molecules We find that the rate of formation and the stability of the unwound complex depend profoundly on supercoiling and that supercoiling exerts its effects mechanically (through torque), and not structurally (through the number and position of supercoils) The approach should permit analysis of other nucleic-acid-processing factors that cause changes in DNA twist and/or DNA compaction
TL;DR: This work shows that the Peyrard-Bishop nonlinear dynamical model of DNA gives an accurate representation of the instability profile of a defined sequence of DNA, as verified using S1 nuclease cleavage assays.
Abstract: It has long been known that double-stranded DNA is subject to temporary, localized openings of its two strands. Particular regions along a DNA polymer are destabilized structurally by available thermal energy in the system. The localized sequence of DNA determines the physical properties of a stretch of DNA, and that in turn determines the opening profile of that DNA fragment. We show that the Peyrard-Bishop nonlinear dynamical model of DNA, which has been used to simulate denaturation of short DNA fragments, gives an accurate representation of the instability profile of a defined sequence of DNA, as verified using S1 nuclease cleavage assays. By comparing results for a non-promoter DNA fragment, the adenovirus major late promoter, the adeno-associated viral P5 promoter and a known P5 mutant promoter that is inactive for transcription, we show that the predicted openings correlate almost exactly with the promoter transcriptional start sites and major regulatory sites. Physicists have speculated that localized melting of DNA might play a role in gene transcription and other processes. Our data link sequence-dependent opening behavior in DNA to transcriptional activity for the first time.
TL;DR: It is proposed that Pol12, the B subunit of the DNA polymerase alpha (Pol1)-primase complex, plays a key role in linking telomerase action with the completion of lagging strand synthesis, and in a regulatory step required for telomere capping.
Abstract: The regulation of telomerase action, and its coordination with conventional DNA replication and chromosome end "capping," are still poorly understood. Here we describe a genetic screen in yeast for mutants with relaxed telomere length regulation, and the identification of Pol12, the B subunit of the DNA polymerase alpha (Pol1)-primase complex, as a new factor involved in this process. Unlike many POL1 and POL12 mutations, which also cause telomere elongation, the pol12-216 mutation described here does not lead to either reduced Pol1 function, increased telomeric single-stranded DNA, or a reduction in telomeric gene silencing. Instead, and again unlike mutations affecting POL1, pol12-216 is lethal in combination with a mutation in the telomere end-binding and capping protein Stn1. Significantly, Pol12 and Stn1 interact in both two-hybrid and biochemical assays, and their synthetic-lethal interaction appears to be caused, at least in part, by a loss of telomere capping. These data reveal a novel function for Pol12 and a new connection between DNA polymerase alpha and Stn1. We propose that Pol12, together with Stn1, plays a key role in linking telomerase action with the completion of lagging strand synthesis, and in a regulatory step required for telomere capping.
TL;DR: It is shown that AID efficiently deaminates C on both DNA strands of a supercoiled plasmid, acting preferentially on SHM hotspot motifs, suggesting a mechanism of AID targeting in vivo.
Abstract: The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination of Ig genes. It has been shown that in vitro, AID protein deaminates C in single-stranded DNA or the coding-strand DNA that is being transcribed but not in double-stranded DNA. However, in vivo, both DNA strands are mutated equally during SHM. We show that AID efficiently deaminates C on both DNA strands of a supercoiled plasmid, acting preferentially on SHM hotspot motifs. However, this DNA is not targeted by AID when it is relaxed after treatment with topoisomerase I, and thus, supercoiling plays a crucial role for AID targeting to this DNA. Most of the mutations are in negatively supercoiled regions, suggesting a mechanism of AID targeting in vivo. During transcription the DNA sequences upstream of the elongating RNA polymerase are negatively supercoiled, and this transient change in DNA topology may allow AID to access both DNA strands.
TL;DR: The 2.8 Å structure of the bacteriophage RB69 replicative DNA polymerase attempting to process an abasic site analog is reported, indicating that the different conformations observed in the crystal may be part of the active site switching reaction pathway.
Abstract: Abasic sites are common DNA lesions, which are strong blocks to replicative polymerases and are potentially mutagenic when bypassed. We report here the 2.8 A structure of the bacteriophage RB69 replicative DNA polymerase attempting to process an abasic site analog. Four different complexes were captured in the crystal asymmetric unit: two have DNA in the polymerase active site whereas the other two molecules are in the exonuclease mode. When compared to complexes with undamaged DNA, the DNA surrounding the abasic site reveals distinct changes suggesting why the lesion is so poorly bypassed: the DNA in the polymerase active site has not translocated and is therefore stalled, precluding extension. All four molecules exhibit conformations that differ from the previously published structures. The polymerase incorporates dAMP across the lesion under crystallization conditions, indicating that the different conformations observed in the crystal may be part of the active site switching reaction pathway.