Although genome analyses have suggested parallels between archaeal and eukaryotic replication systems, little is known about the DNA replication mechanism in Archaea. By two-dimensional gel electrophoreses we positioned a replication origin (oriC) within 1 kb in the chromosomal DNA of Pyrococcus abyssi, an anaerobic hyperthermophile, and demonstrated that the oriC is physically linked to the cdc6 gene. Our chromatin immunoprecipitation assays indicated that P. abyssi Cdc6 and minichromosome maintenance (MCM) proteins bind preferentially to the oriC region in the exponentially growing cells. Whereas the oriC association of MCM was specifically inhibited by stopping DNA replication with puromycin treatment, Cdc6 protein stayed bound to the replication origin after de novo protein synthesis was inhibited. Our data suggest that archaeal and eukaryotic Cdc6 and MCM proteins function similarly in replication initiation and imply that an oriC association of MCM could be regulated by an unknown mechanism in Archaea.Archaea ͉ minichromosome maintenance helicase ͉ origin association ͉ initiator protein T he initiation step of chromosomal DNA replication is of fundamental importance for the inheritance of genetic material and cell cycle regulation and therefore has attracted considerable experimental interest over the years. Detailed studies of DNA replication initiation in Eukarya and Bacteria have identified multistep processes leading to the unwinding of duplex DNA at a replication origin (recently reviewed in refs. 1 and 2). In comparison with the relatively detailed knowledge obtained for Bacteria and Eukarya with regard to their replication initiation, until recently, no data were available on the replication mechanism in Archaea, the third domain of life. Because many eukaryotic replication proteins, including the initiator proteins Orc1, Cdc6, and minichromosome maintenance (MCM), have homologs in Archaea but not in Bacteria (3-6), studies of the archaeal replication system have the potential to elucidate hitherto unresolved questions with regard to the eukaryotic replication mechanism. In the absence of biochemical or genetic in vivo data, whether the archaeal initiator proteins are functionally analogous to their eukaryotic counterparts has remained elusive. Nevertheless, the recent demonstration that the single MCM protein of the archaeon Methanobacterium thermoautotrophicum forms a double-homohexamer complex with a processive 3Ј to 5Ј ATP-dependent DNA helicase activity in vitro (7-9) has illustrated how identifying the properties of an archaeal replication protein can aid in understanding processes leading to unwinding of the replication origin by eukaryotic replication proteins.For a long time, the study of archaeal DNA replication in vivo and in vitro has been hampered by the absence of information about the nature of the replication origins in these atypical prokaryotes. Complete archaeal genome sequences have made it possible to predict the location of a single replication origin in M. thermoautotrophicum ...
The initiator protein (RepE) of F factor, a plasmid involved in sexual conjugation in Escherichia coli, has dual functions during the initiation of DNA replication which are determined by whether it exists as a dimer or as a monomer. A RepE monomer functions as a replication initiator, but a RepE dimer functions as an autogenous repressor. We have solved the crystal structure of the RepE monomer bound to an iteron DNA sequence of the replication origin of plasmid F. The RepE monomer consists of topologically similar N-and C-terminal domains related to each other by internal pseudo 2-fold symmetry, despite the lack of amino acid similarities between the domains. Both domains bind to the two major grooves of the iteron (19 bp) with different binding affinities. The C-terminal domain plays the leading role in this binding, while the N-terminal domain has an additional role in RepE dimerization. The structure also suggests that superhelical DNA induced at the origin of plasmid F by four RepEs and one HU dimer has an essential role in the initiation of DNA replication.
Pyrococcus furiosus, a hyperthermophilic Archaea, has homologs of the eukaryotic MCM (mini-chromosome maintenance) helicase and GINS complex. The MCM and GINS proteins are both essential factors to initiate DNA replication in eukaryotic cells. Many biochemical characterizations of the replication-related proteins have been reported, but it has not been proved that the homologs of each protein are also essential for replication in archaeal cells. Here, we demonstrated that the P. furiosus GINS complex interacts with P. furiosus MCM. A chromatin immunoprecipitation assay revealed that the GINS complex is detected preferentially at the oriC region on Pyrococcus chromosomal DNA during the exponential growth phase but not in the stationary phase. Furthermore, the GINS complex stimulates both the ATPase and DNA helicase activities of MCM in vitro. These results strongly suggest that the archaeal GINS is involved in both the initiation and elongation processes of DNA replication in P. furiosus, as observed in eukaryotic cells.
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 welldefined 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. EMBO reports 4, 154-158 (2003)
Replication of mini-F plasmids requires the initiator protein RepE, which binds specifically to four iterons within the origin (ori2), as well as some host factors that are involved in chromosomal DNA replication. To understand the role of host factors and RepE in the early steps of mini-F DNA replication, we examined the effects of RepE and the Escherichia coli proteins DnaA and HU on the localized melting of ori2 DNA in a purified in vitro system. We found that the binding of RepE to an iteron causes a 50 degrees bend at or around the site of binding. RepE and HU exhibited synergistic effects on the localized melting within the ori2 region, as detected by sensitivity to the single-strand specific P1 endonuclease. This opening of duplex DNA occurred around the 13mer of ori2, whose sequence closely resembles the set of 13mers found in the chromosomal origin oriC. Further addition of DnaA to the reaction mixture increased the efficiency of melting and appeared to extend melting to the adjacent AT-rich region. Moreover, DNA melting with appreciably higher efficiencies was observed with mutant forms of RepE that were previously shown to be hyperactive both in DNA binding in vitro and in initiator activity in vivo. We propose that the binding of RepE to four iterons of ori2 causes bending at the sites of RepE binding and, with the assistance of HU, induces a localized melting in the 13mer region. The addition of DnaA extends melting to the AT-rich region, which could then serve as the entry site for the DnaB-DnaA complex, much as has been documented for oriC dependent replication.
The origin of DNA replication (oriC) of the hyperthermophilic archaeon Pyrococcus abyssi contains multiple ORB and mini-ORB repeats that show sequence similarities to other archaeal ORB (origin recognition box). We report here that the binding of Cdc6/Orc1 to a 5 kb region containing oriC in vivo was highly specific both in exponential and stationary phases, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip). The oriC region is practically the sole binding site for the Cdc6/Orc1, thereby distinguishing oriC in the 1.8 M bp genome. We found that the 5 kb region contains a previously unnoticed cluster of ORB and mini-ORB repeats in the gene encoding the small subunit (dp1) for DNA polymerase II (PolD). ChIP and the gel retardation analyses further revealed that Cdc6/Orc1 specifically binds both of the ORB clusters in oriC and dp1. The organization of the ORB clusters in the dp1 and oriC is conserved during evolution in the order Thermococcales, suggesting a role in the initiation of DNA replication. Our ChIP-chip analysis also revealed that Mcm alters the binding specificity to the oriC region according to the growth phase, consistent with its role as a licensing factor.
The RepE initiator protein (251 residues) is essential for mini-F replication in Escherichia coli and exhibits two major functions: initiation of DNA replication from ori2 and autogenous repression of repE transcription. Whereas the initiation is mediated by RepE monomers that bind to the ori2 iterons (direct repeats), the autogenous repression is mediated by dimers that bind to the repE operator, which contains an inverted repeat sequence related to the iterons. We now report that the binding of RepE to these DNA sites is primarily determined by the C-terminal region of this protein. The mutant RepE proteins lacking either the N-terminal 33 (or more) residues or the C-terminal 7 (or more) residues were first shown to be defective in binding to both the ori2 and the operator DNAs. However, direct screening and analysis of mutant RepEs which are specifically affected in binding to the ori2 iterons revealed that the mutations (mostly amino acid substitutions) occur exclusively in the C-terminal region (residues 168 to 242). These mutant proteins exhibited reduced binding to ori2 and no detectable binding to the operator. Thus, whereas truncation of either end of RepE can destroy the DNA-binding activities, the C-terminal region appears to represent a primary DNA-binding domain of RepE for both ori2 and the operator. Analogous DNA-binding domains seem to be conserved among the initiator proteins of certain related plasmids.The mini-F plasmid, like the parental F factor, is stably maintained in Escherichia coli with one or two copies per host chromosome. Replication of mini-F requires several host factors, including DnaA (14,22,31), HU (34,38), and a subset of heat shock proteins (DnaK, DnaJ, and GrpE) (6, 18), besides the plasmid-encoded replication initiator protein, RepE (251 residues, 29 kDa) (see reference 21). A minimal mini-F consists of an origin of replication (ori2), repE, and a drug resistance gene such as bla and exhibits high copy numbers (10 to 15 copies per chromosome) (19) due to the lack of incC, which negatively modulates the copy number. The ori2 region contains two DnaA boxes, an AT-rich region, and four direct repeats (iterons) of 19 bp, whereas the repE operator contains an inverted repeat whose half sequence (10 bp) resembles the ori2 iterons (8-bp matches) (Fig. 1). The RepE protein, a sequence-specific DNA-binding protein, binds to the ori2 iterons and to the operator (27,36). The binding to these separate but related DNA sequences plays a key role in the regulation of mini-F replication: RepE acts as an initiator of DNA replication through binding to ori2 and as an autogenous repressor of repE transcription through binding to the operator (see references 21, 30, and 37).We recently reported that the two functions of RepE are carried out by structurally distinct forms of the protein. One of the RepE mutants (RepE54), previously selected for their ability to replicate in the dnaJ mutant host (16), produced hyperactive RepE that cannot form dimers (17), unlike the wild-type protein that is fou...
The initiation step is a key process to regulate the frequency of DNA replication. Although recent studies in Archaea defined the origin of DNA replication (oriC) and the Cdc6/Orc1 homolog as an origin recognition protein, the location and mechanism of duplex opening have remained unclear. We have found that Cdc6/Orc1 binds to oriC and unwinds duplex DNA in the hyperthermophilic archaeon Pyrococcus furiosus, by means of a P1 endonuclease assay. A primer extension analysis further revealed that this localized unwinding occurs in the oriC region at a specific site, which is 12-bp long and rich in adenine and thymine. This site is different from the predicted duplex unwinding element (DUE) that we reported previously. We also discovered that Cdc6/Orc1 induces topological changes in supercoiled oriC DNA, and that this process is dependent on the AAA+ domain. These results indicate that topological alterations of oriC DNA by Cdc6/Orc1 introduce a single-stranded region at the 12-mer site, that could possibly serve as an entry point for Mcm helicase.
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