DNA Unwinding Element Research Articles

DNA stretching by bacterial initiators promotes replication origin opening

SummaryMany replication initiators form higher-order oligomers that process host origins to promote replisome formation. In addition to dedicated duplex DNA-binding domains, cellular initiators possess AAA+ (ATPases Associated with various cellular Activities) elements that drive functions ranging from protein assembly to origin recognition. In bacteria, the AAA+ domain of the initiator DnaA has been suggested to bind single-stranded DNA formed during origin melting. Here we show crystallographically and in solution that the ATP-dependent assembly of DnaA into a spiral oligomer creates a continuous surface that allows successive AAA+ domains to bind and extend single-stranded DNA segments. The mechanism of binding is unexpectedly similar to that of RecA, a homologous recombination factor, but it differs in that DnaA promotes a nucleic acid conformation that prevents pairing of a complementary strand. These findings, combined with strand-displacement assays, indicate that DnaA melts replication origins by a direct ATP-dependent stretching mechanism. Comparative studies reveal remarkable commonalities between the approach used by DnaA to engage DNA substrates and other, nucleic acid-dependent AAA+ systems.

Expansions, contractions, and fragility of the spinocerebellar ataxia type 10 pentanucleotide repeat in yeast

Spinocerebellar ataxia 10 (SCA10) is an autosomal dominant disease caused by large-scale expansions of the (ATTCT)(n) repeat within an intron of the human ATXN10 gene. In contrast to other expandable repeats, this pentanucleotide repeat does not form stable intra- or interstranded DNA structures, being a DNA unwinding element instead. We analyzed the instability of the (ATTCT)(n) repeat in a yeast experimental system, where its expansions led to inactivation of the URA3 reporter gene. The inactivation was due to a dramatic decrease in the mRNA levels owing to premature transcription termination and RNA polyadenylation at the repeat. The rates of expansions strongly increased with the repeat's length, mimicking genetic anticipation in human pedigrees. A first round of genetic analysis showed that a functional TOF1 gene precludes, whereas a functional RAD5 gene promotes, expansions of the (ATTCT)(n) repeat. We hypothesize that repeat expansions could occur upon fortuitous template switching during DNA replication. The rate of repeat contractions was elevated in the Tof1 knockout strain, but it was not affected by the RAD5 gene. Supporting the notion of replication irregularities, we found that (ATTCT)(n) repeats also cause length-dependent chromosomal fragility in yeast. Repeat-mediated fragility was also affected by the Tof1 and Rad5 proteins, being reduced in their absence.

Origin Remodeling and Opening in Bacteria Rely on Distinct Assembly States of the DnaA Initiator

The initiation of DNA replication requires the melting of chromosomal origins to provide a template for replisomal polymerases. In bacteria, the DnaA initiator plays a key role in this process, forming a large nucleoprotein complex that opens DNA through a complex and poorly understood mechanism. Using structure-guided mutagenesis, biochemical, and genetic approaches, we establish an unexpected link between the duplex DNA-binding domain of DnaA and the ability of the protein to both self-assemble and engage single-stranded DNA in an ATP-dependent manner. Intersubunit cross-talk between this domain and the DnaA ATPase region regulates this link and is required for both origin unwinding in vitro and initiator function in vivo. These findings indicate that DnaA utilizes at least two different oligomeric conformations for engaging single- and double-stranded DNA, and that these states play distinct roles in controlling the progression of initiation.

The DNA Unwinding Element Binding Protein DUE-B Interacts with Cdc45 in Preinitiation Complex Formation

Template unwinding during DNA replication initiation requires the loading of the MCM helicase activator Cdc45 at replication origins. We show that Cdc45 interacts with the DNA unwinding element (DUE) binding protein DUE-B and that these proteins localize to the DUEs of active replication origins. DUE-B and Cdc45 are not bound at the inactive c-myc replicator in the absence of a functional DUE or at the recently identified ataxin 10 (ATX10) origin, which is silent before disease-related (ATTCT)(n) repeat length expansion of its DUE sequence, despite the presence of the origin recognition complex (ORC) and MCM proteins at these origins. Addition of a heterologous DUE to the ectopic c-myc origin, or expansion of the ATX10 DUE, leads to origin activation, DUE-B binding, and Cdc45 binding. DUE-B, Cdc45, and topoisomerase IIbeta binding protein 1 (TopBP1) form complexes in cell extracts and when expressed from baculovirus vectors. During replication in Xenopus egg extracts, DUE-B and Cdc45 bind to chromatin with similar kinetics, and DUE-B immunodepletion blocks replication and the loading of Cdc45 and a fraction of TopBP1. The coordinated binding of DUE-B and Cdc45 to origins and the physical interactions of DUE-B, Cdc45, and TopBP1 suggest that complexes of these proteins are necessary for replication initiation.

DiaA Dynamics Are Coupled with Changes in Initial Origin Complexes Leading to Helicase Loading

Chromosomal replication initiation requires the regulated formation of dynamic higher order complexes. Escherichia coli ATP-DnaA forms a specific multimer on oriC, resulting in DNA unwinding and DnaB helicase loading. DiaA, a DnaA-binding protein, directly stimulates the formation of ATP-DnaA multimers on oriC and ensures timely replication initiation. In this study, DnaA Phe-46 was identified as the crucial DiaA-binding site required for DiaA-stimulated ATP-DnaA assembly on oriC. Moreover, we show that DiaA stimulation requires only a subgroup of DnaA molecules binding to oriC, that DnaA Phe-46 is also important in the loading of DnaB helicase onto the oriC-DnaA complexes, and that this process also requires only a subgroup of DnaA molecules. Despite the use of only a DnaA subgroup, DiaA inhibited DnaB loading on oriC-DnaA complexes, suggesting that DiaA and DnaB bind to a common DnaA subgroup. A cellular factor can relieve the DiaA inhibition, allowing DnaB loading. Consistently, DnaA F46A caused retarded initiations in vivo in a DiaA-independent manner. It is therefore likely that DiaA dynamics are crucial in the regulated sequential progress of DnaA assembly and DnaB loading. We accordingly propose a model for dynamic structural changes of initial oriC complexes loading DiaA or DnaB helicase.

The splicing factor SF2/ASF binds to ARS homologs in a human rDNA replication origin

The function of the relatively well-studied DNA replication origins in the yeast Saccharomyces cerevisiae is dependent upon interactions between origin replication complex (ORC) proteins and several defined origin sequence elements, including the 11 bp ARS consensus sequence (ACS). Although the ORC proteins, as well as numerous other protein components required for DNA replication initiation, are largely conserved between yeast and mammals, DNA sequences within mammalian replication origins are highly variable and sequences homologous to the yeast ACS elements are generally not present. We have previously identified several replication initiation sites within the nontranscribed spacer region of the human ribosomal RNA gene, and found that two highly utilized sites each contain a homologue of the yeast ACS embedded within a DNA unwinding element and a matrix attachment region. Here we examine protein binding within these initiation sites, and demonstrate that these ACS homologues specifically bind the alternate splicing factor SF2/ASF as well as GAPDH in vitro, and present evidence that the SF2/ASF interaction also occurs within the nuclei of intact cells. As the moderate upregulation of SF2/ASF has been linked to oncogenesis through the promotion of alternatively spliced forms of several regulatory proteins, our results suggest an additional mechanism by which SF2/ASF may influence the transformed cell phenotype.

A Common Mechanism for the ATP-DnaA-dependent Formation of Open Complexes at the Replication Origin

Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATP-specific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.

Unstable Spinocerebellar Ataxia Type 10 (ATTCT)·(AGAAT) Repeats Are Associated with Aberrant Replication at the ATX10 Locus and Replication Origin-Dependent Expansion at an Ectopic Site in Human Cells

Spinocerebellar ataxia type 10 (SCA10) is associated with expansion of (ATTCT)n repeats (where n is the number of repeats) within the ataxin 10 (ATX10/E46L) gene. The demonstration that (ATTCT)n tracts can act as DNA unwinding elements (DUEs) in vitro has suggested that aberrant replication origin activity occurs at expanded (ATTCT)n tracts and may lead to their instability. Here, we confirm these predictions. The wild-type ATX10 locus displays inefficient origin activity, but origin activity is elevated at the expanded ATX10 loci in patient-derived cells. To test whether (ATTCT)n tracts can potentiate origin activity, cell lines were constructed that contain ectopic copies of the c-myc replicator in which the essential DUE was replaced by ATX10 DUEs with (ATTCT)n. ATX10 DUEs containing (ATTCT)27 or (ATTCT)48, but not (ATTCT)8 or (ATTCT)13, could substitute functionally for the c-myc DUE, but (ATTCT)48 could not act as an autonomous replicator. Significantly, chimeric c-myc replicators containing ATX10 DUEs displayed length-dependent (ATTCT)n instability. By 250 population doublings, dramatic two- and fourfold length expansions were observed for (ATTCT)27 and (ATTCT)48 but not for (ATTCT)8 or (ATTCT)13. These results implicate replication origin activity as one molecular mechanism associated with the instability of (ATTCT)n tracts that are longer than normal length.

Structure and Function of the c-myc DNA-unwinding Element-binding Protein DUE-B

Local zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNA-unwinding element-binding protein DUE-B in vivo. We present here the 2.0A crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both D-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA.

Differential Binding of Replication Proteins across the Human c-myc Replicator

The binding of the prereplication complex proteins Orc1, Orc2, Mcm3, Mcm7, and Cdc6 and the novel DNA unwinding element (DUE) binding protein DUE-B to the endogenous human c-myc replicator was studied by chromatin immunoprecipitation. In G(1)-arrested HeLa cells, Mcm3, Mcm7, and DUE-B were prominent near the DUE, while Orc1 and Orc2 were least abundant near the DUE and more abundant at flanking sites. Cdc6 binding mirrored that of Orc2 in G(1)-arrested cells but decreased in asynchronous or M-phase cells. Similarly, the signals from Orc1, Mcm3, and Mcm7 were at background levels in cells arrested in M phase, whereas Orc2 retained the distribution seen in G(1)-phase cells. Previously shown to cause histone hyperacetylation and delocalization of replication initiation, trichostatin A treatment of cells led to a parallel qualitative change in the distribution of Mcm3, but not Orc2, across the c-myc replicator. Orc2, Mcm3, and DUE-B were also bound at an ectopic c-myc replicator, where deletion of sequences essential for origin activity was associated with the loss of DUE-B binding or the alteration of chromatin structure and loss of Mcm3 binding. These results show that proteins implicated in replication initiation are selectively and differentially bound across the c-myc replicator, dependent on discrete structural elements in DNA or chromatin.

Multiple Initiation Sites within the Human Ribosomal RNA Gene

Numerous studies have demonstrated that DNA replication initiates within the 30 kBnontranscribed spacer (NTS) region of the human ribosomal RNA gene (rDNA). Using a series ofclosely spaced primer pairs to measure nascent leading strand abundance in mid and late S phasecells isolated by centrifugal elutriation, we find evidence for one highly preferred initiation site andtwo less utilized sites within a 6 kb region of the NTS. The initiation sites co-localize withsignificant DNA unwinding elements (DUEs), matrix attachment regions (MARs), and ARS-likesequences. An intrinsic DNA bending site was localized by circular permutation analysis to withinseveral hundred base pairs of one initiation site. While DUE and MAR elements occur elsewherethroughout the 43 kb rDNA sequence, the close association of DUE and MAR elements occursonly near replication initiation sites, a juxtaposition also seen in other well-studied mammalianreplication initiation sites. The utilization of rDNA initiation sites close to DUE and MAR elementsin mid and late S phase, but not in very early S phase as previously shown, suggests that in rRNAgenes, contributions from these sequence-associated properties may be more significant to initiationsites associated with transcriptionally inactive genes than to initiation sites associated with activegenes.

Advances in mechanisms of genetic instability related to hereditary neurological diseases

Substantial progress has been realized in the past several years in our understanding of the molecular mechanisms responsible for the expansions and deletions (genetic instabilities) of repeating tri-, tetra- and pentanucleotide repeating sequences associated with a number of hereditary neurological diseases. These instabilities occur by replication, recombination and repair processes, probably acting in concert, due to slippage of the DNA complementary strands relative to each other. The biophysical properties of the folded-back repeating sequence strands play a critical role in these instabilities. Non-B DNA structural elements (hairpins and slipped structures, DNA unwinding elements, tetraplexes, triplexes and sticky DNA) are described. The replication mechanisms are influenced by pausing of the replication fork, orientation of the repeat strands, location of the repeat sequences relative to replication origins and the flap endonuclease. Methyl-directed mismatch repair, nucleotide excision repair, and repair of damage caused by mutagens are discussed. Genetic recombination and double-strand break repair advances in Escherichia coli, yeast and mammalian models are reviewed. Furthermore, the newly discovered capacities of certain triplet repeat sequences to cause gross chromosomal rearrangements are discussed.

Base Pair Opening in Three DNA-unwinding Elements

DNA-unwinding elements are specific base sequences that are located in the origin of DNA replication where they provide the start point for strand separation and unwinding of the DNA double helix. In the present work we have obtained the first characterization of the opening of individual base pairs in DNA-unwinding elements. The three DNA molecules investigated reproduce the 13-mer DNA-unwinding elements present in the Escherichia coli chromosome. The base sequences of the three 13-mers are conserved in the origins of replication of enteric bacterial chromosomes. The exchange of imino protons with solvent protons was measured for each DNA as a function of the concentration of exchange catalyst using nuclear magnetic resonance spectroscopy. The exchange rates provided the rates and the equilibrium constants for opening of individual base pairs in each DNA at 20 degrees C. The results reveal that the kinetics and energetics of the opening reactions for AT/TA base pairs are different in the three DNA-unwinding elements due to long range effects of the base sequence. These differences encompass the AT/TA base pairs that are conserved in various bacterial genomes. Furthermore, a qualitative correlation is observed between the kinetics and energetics of opening of AT/TA base pairs and the location of the corresponding DNA-unwinding element in the origin of DNA replication.

The synthesis and initial characterization of an immobilized DNA unwinding element binding (DUE-B) protein chromatographic stationary phase

The DNA unwinding element binding protein (DUE-B) plays a key role in DNA replication. The DUE-B protein has been immobilized on a liquid chromatography support and the resulting immobilized protein column was used for the on-line screening of a series of steroids. The DUE-B protein was expressed with an added C-terminal sequence of six adjacent histidine residues, a His 6-tag and immobilized on a chiral ligand exchange support, the CLC-L column, using Ni 2+ as the coordinating metal ion. The chromatographic retentions of 12 steroids were determined on the DUE-B/CLC-L column. The magnitudes of the steroid-immobilized DUE-B interactions, reflected by the observed retention times, correlated to the effect of the steroids in the cell-free replication system, i.e. the longer the retention, the greater the increase in DNA replication. The coefficient of determination for the %DNA activities linear relation to retention time was 0.9694. The data suggest that the DUE-B/CLC-L phase can be used for on-line pharmacological studies. The results also indicated that His-tagged proteins can be directly immobilized on the CLC-L stationary phase and the resulting columns used as rapid screens for the isolation and identification of small molecule or protein ligands from complex biological or chemical mixtures.

The c-myc DNA-unwinding Element-binding Protein Modulates the Assembly of DNA Replication Complexes in Vitro

The presence of DNA-unwinding elements (DUEs) at eukaryotic replicators has raised the question of whether these elements contribute to origin activity by their intrinsic helical instability, as protein-binding sites, or both. We used the human c-myc DUE as bait in a yeast one-hybrid screen and identified a DUE-binding protein, designated DUE-B, with a predicted mass of 23.4 kDa. Based on homology to yeast proteins, DUE-B was previously classified as an aminoacyl-tRNA synthetase; however, the human protein is approximately 60 amino acids longer than its orthologs in yeast and worms and is primarily nuclear. In vivo, chromatin-bound DUE-B localized to the c-myc DUE region. DUE-B levels were constant during the cell cycle, although the protein was preferentially phosphorylated in cells arrested early in S phase. Inhibition of DUE-B protein expression slowed HeLa cell cycle progression from G1 to S phase and induced cell death. DUE-B extracted from HeLa cells or expressed from baculovirus migrated as a dimer during gel filtration and co-purified with ATPase activity. In contrast to endogenous DUE-B, baculovirus-expressed DUE-B efficiently formed high molecular mass complexes in Xenopus egg and HeLa extracts. In Xenopus extracts, baculovirus-expressed DUE-B inhibited chromatin replication and replication protein A loading in the presence of endogenous DUE-B, suggesting that differential covalent modification of these proteins can alter their effect on replication. Recombinant DUE-B expressed in HeLa cells restored replication activity to egg extracts immunodepleted with anti-DUE-B antibody, suggesting that DUE-B plays an important role in replication in vivo.

Chemical footprinting of structural and functional elements of dhfr oriβ during the CHOC 400 cell cycle

Oriβ, an origin of replication 3′ to Chinese hamster dihydrofolate reductase ( dhfr) gene, contains several sequence elements that function as components of a chromosomal replicator. Here we have examined sensitivity to KMnO 4 in vitro and in living cells of three regions within dhfr oriβ which contribute to replicator function: the origin of bidirectional DNA replication (OBR) that serves as an initiation site for DNA synthesis, a stably bent DNA region that binds activator protein one (AP-1) and RIP60 in vitro, and an AT-rich region that contains a dA/dT 23 dinucleotide repeat that has properties of a DNA unwinding element. The in vitro patterns of KMnO 4 modification in linear plasmid differed from that in supercoiled plasmid most prominently in the dA/dT 23 repeat, with evidence of palindrome extrusion in supercoiled plasmid. Although palindrome extrusion was not detected in genomic DNA during the cell cycle, the pattern of genomic DNA modification within the dA/dT 23 repeat differed substantially from that of either linear or plasmid DNA in vitro. An AT-rich region that borders the dA/dT repeat was also highly sensitive to modification by KMnO 4 in cells. Within the bent DNA region, the patterns of chemical modification of both the AP-1 and RIP60 sites differed between plasmid and genomic DNA, and minor differences in the in vitro and cellular modification patterns also were observed for the OBR. Nonetheless, there was little evidence of cell cycle-specific modifications in any sequence examined. These studies suggest that sequences within dhfr oriβ adopt specific conformations in cells, with the most prominent changes in the AT-rich region associated with the dA/dT 23 repeat and DNA unwinding.

PATTERNFINDER: combined analysis of DNA regulatory sequences and double-helix stability

BackgroundRegulatory regions that function in DNA replication and gene transcription contain specific sequences that bind proteins as well as less-specific sequences in which the double helix is often easy to unwind. Progress towards predicting and characterizing regulatory regions could be accelerated by computer programs that perform a combined analysis of specific sequences and DNA unwinding properties.ResultsHere we present PATTERNFINDER, a web server that searches DNA sequences for matches to specific or flexible patterns, and analyzes DNA helical stability. A batch mode of the program generates a tabular map of matches to multiple, different patterns. Regions flanking pattern matches can be targeted for helical stability analysis to identify sequences with a minimum free energy for DNA unwinding. As an example application, we analyzed a regulatory region of the human c-myc proto-oncogene consisting of a single-strand-specific protein binding site within a DNA region that unwindsin vivo. The predicted region of minimal helical stability overlapped both the protein binding site and the unwound DNA region identified experimentally.ConclusionsThe PATTERNFINDER web server permits localization of known functional elements or landmarks in DNA sequences as well as prediction of potential new elements. Batch analysis of multiple patterns facilitates the annotation of DNA regulatory regions. Identifying specific pattern matches linked to DNA with low helical stability is useful in characterizing regulatory regions for transcription, replication and other processes and may predict functional DNA unwinding elements.PATTERNFINDER can be accessed freely at:

Multiple functional elements comprise a Mammalian chromosomal replicator.

The structure of replication origins in metazoans is only nominally similar to that in model organisms, such as Saccharomyces cerevisiae. By contrast to the compact origins of budding yeast, in metazoans multiple elements act as replication start sites or control replication efficiency. We first reported that replication forks diverge from an origin 5' to the human c-myc gene and that a 2.4-kb core fragment of the origin displays autonomous replicating sequence activity in plasmids and replicator activity at an ectopic chromosomal site. Here we have used clonal HeLa cell lines containing mutated c-myc origin constructs integrated at the same chromosomal location to identify elements important for DNA replication. Replication activity was measured before or after integration of the wild-type or mutated origins using PCR-based nascent DNA abundance assays. We find that deletions of several segments of the c-myc origin, including the DNA unwinding element and transcription factor binding sites, substantially reduced replicator activity, whereas deletion of the c-myc promoter P1 had only a modest effect. Substitution mutagenesis indicated that the sequence of the DNA unwinding element, rather than the spacing of flanking sequences, is critical. These results identify multiple functional elements essential for c-myc replicator activity.

Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin.

Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria. In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo. We have detected, in two main archaeal phyla, short RNA-primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments. Mapping of replication initiation points further showed that discontinuous DNA replication in P. abyssi starts at a well-defined site within the oriC recently identified in this hyperthermophile. Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea. Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.

Specific recognition of the replication origins of the kinetoplast DNA.

The understanding of the process of chromosome replication initiation and its control in cells and subcellular organelle requires the identification and characterization of origins of replication and other specific control elements in the DNA template, as well as the counterpart proteins that interact with these regulatory sequences. An early event that had been described during replication initiation of bacterial chromosomes, plasmids, bacterial and animal viruses and lower eukaryotes, is the recognition of a unique sequence at the replication origin, known as the origin recognition element (ORE), by a special origin binding protein and the binding of the protein onto this site. This protein–DNA interaction is a key event in chromosome replication initiation. It results in the local, non-catalytic melting of the nearby DNA unwinding element (DUE), a sequence with a relatively lower melting profile, enabling the loading of DNA helicases and the catalytic unwinding of the origin site. Furthermore, binding of the origin binding protein directs, through specific protein–protein interactions, other replication initiation proteins onto the origin site to be assembled into a functional replication initiation complex (reviewed in [1–8]). We are studying these early events in the replication of DNA in eukaryotes, using the trypanosomatid protozoan Crithidia fasciculata as an experimental model. We focus our attention on the replication of a unique extra-chromosomal DNA