Interaction of fission yeast ORC with essential adenine/thymine stretches in replication origins.

BACKGROUND: Eukaryotic DNA replication is initiated from distinct regions on the chromosome. However, the mechanism for recognition of replication origins is not known for most eukaryotes. In fission yeast, replication origins are isolated as autonomously replicating sequences (ARSs). Multiple adenine/thymine clusters are essential for replication, but no short consensus sequences are found. In this paper, we examined the interaction of adenine/thymine clusters with the replication initiation factor ORC. RESULTS: The SpOrc1 or SpOrc2 immunoprecipitates (IPs) containing at least four subunits of SpORC, interacted with the ars2004 fragment, which is derived from a predominant replication origin on the chromosome. SpORC-IPs preferentially interacted with two regions of the ars2004, which consist of consecutive adenines and AAAAT repeats and are essential for ARS activity. The nucleotide sequences required for the interaction with SpORC-IPs correspond closely to those necessary for in vivo ARS activity. CONCLUSION: Our results suggest that the SpORC interacts with adenine/thymine stretches, which have been shown to be the most important component in the fission yeast replication origin. The presence of multiple SpORC-binding sites, with certain sequence variations, is characteristic for the fission yeast replication origins.

Association of fission yeast Orp1 and Mcm6 proteins with chromosomal replication origins.

We have previously shown that replication of fission yeast chromosomes is initiated in distinct regions. Analyses of autonomous replicating sequences have suggested that regions required for replication are very different from those in budding yeast. Here, we present evidence that fission yeast replication origins are specifically associated with proteins that participate in initiation of replication. Most Orp1p, a putative subunit of the fission yeast origin recognition complex (ORC), was found to be associated with chromatin-enriched insoluble components throughout the cell cycle. In contrast, the minichromosome maintenance (Mcm) proteins, SpMcm2p and SpMcm6p, encoded by the nda1(+)/cdc19(+) and mis5(+) genes, respectively, were associated with chromatin DNA only during the G(1) and S phases. Immunostaining of spread nuclei showed SpMcm6p to be localized at discrete foci on chromatin during the G(1) and S phases. A chromatin immunoprecipitation assay demonstrated that Orp1p was preferentially localized at the ars2004 and ars3002 origins of the chromosome throughout the cell cycle, while SpMcm6p was associated with these origins only in the G(1) and S phases. Both Orp1p and SpMcm6p were associated with a 1-kb region that contains elements required for autonomous replication of ars2004. The results suggest that the fission yeast ORC specifically interacts with chromosomal replication origins and that Mcm proteins are loaded onto the origins to play a role in initiation of replication.

Clustered adenine/thymine stretches are essential for function of a fission yeast replication origin.

We have determined functional elements required for autonomous replication of the Schizosaccharomyces pombe ars2004 that acts as an intrinsic chromosomal replication origin. Internal deletion analysis of a 940-bp fragment (ars2004M) showed three regions, I to III, to be required for autonomously replicating sequence (ARS) activity. Eight-base-pair substitutions in the 40-bp region I, composed of arrays of adenines on a DNA strand, resulted in a great reduction of ARS activity. Substitutions of region I with synthetic sequences showed that no specific sequence but rather repeats of three or more consecutive adenines or thymines, without interruption by guanine or cytosine, are required for the ARS activity. The 65-bp region III contains 11 repeats of the AAAAT sequence, while the 165-bp region II has short adenine or thymine stretches and a guanine- and cytosine-rich region which enhances ARS activity. All three regions in ars2004M can be replaced with 40-bp poly(dA/dT) fragments without reduction of ARS activity. Although spacer regions in the ars2004M enhance ARS activity, all could be deleted when an 40-bp poly(dA/dT) fragment was added in place of region I. Our results suggest that the origin activity of fission yeast replicators depends on the number of adenine/thymine stretches, the extent of their clustering, and presence of certain replication-enhancing elements.

Association of autonomous replication activity with replication origins in a human chromosome.

A systematic analysis of the correlation of autonomous replication activity with initiation of replication in a human chromosome was performed. The temporal order of replication of segments in a pericentric 320-kb MEN203 locus on human chromosome 10 (10q11.2) was determined by pulse-labeling of cells with 5-bromodeoxyuridine after synchronization with aphidicolin. The entire MEN203 locus replicated during the late S phase. Two distinct segments replicated earlier than the others in the locus, indicating that replication was initiated within or near these segments. Two other segments also showed an earlier response than the respective neighboring regions. These results suggest that the MEN203 locus contains two distinct replication origins and two possible origins that may be used less frequently. The results were essentially confirmed by synchronization of the cell cycle with mimosine. Analysis of autonomous replication activity of 10-kb long chromosome fragments covering the 320-kb region showed that certain fragments replicated two or three times more efficiently than others. The results are consistent with our previous observations with randomly cloned human chromosome fragments. The replication origins colocalized with fragments exhibiting relatively high autonomous replication activity. Thus, the capacity for autonomous replication of chromosome fragments might be prerequisite for the initiation of chromosomal replication.

Interaction of transcription factor YY1 with a replication-enhancing element, REE1, in an autonomously replicating human chromosome fragment.

We have previously shown that autonomous replication of human chromosome fragments is stimulated by the presence of an 18 bp sequence, REE1, which exhibits transcriptional silencer activity. The REE1 sequence is partly homologous with the serum response element (SRE) required for expression of the human c- fos gene. Here we have examined interaction of REE1 with human nuclear proteins using a gel retardation assay. One of the REE1-protein complexes formed showed almost the same mobility as the SRE-protein complex and complex formation was competitively inhibited by the SRE fragment. The protein complex with REE1 as well as that with SRE was found to contain the transcription factor YY1, known to bind to the SRE. These results suggest that YY1 protein may participate in stimulation of replication through its interaction with REE1.

Identification of a predominant replication origin in fission yeast.

We have identified five autonomously replicating sequences (ARSs) in a 100 kbp region of the Schizosaccharomyces pombe chromosome II. Analyses of replicative intermediates of the chromosome DNA by neutral/neutral two-dimensional gel electrophoresis demonstrated that at least three of these ARS loci operate as chromosomal replication origins. One of the loci,ori2004, was utilized in almost every cell cycle, while the others were used less frequently. The frequency of initiation from the respective chromosomal replication origin was found to be roughly proportional to the efficiency of autonomous replication of the corresponding ARS plasmid. Replication from ori2004 was initiated within a distinct region almost the same as that for replication of the ARS plasmid. These results showed that the ori2004 region of approximately 3 kbp contains all the cis elements essential for initiation of chromosome replication.

A replication-enhancing element with transcriptional silencer activity in autonomously replicating human chromosomal fragments.

We have identified specific nucleotide sequences involved in autonomous replication of human chromosomal fragments in human cells. Nested deletion analysis of a 10.2-kb long human chromosomal fragment showed that replication efficiency of the fragment was reduced to about 50% by loss of a short specific segment. Deletions outside the segment reduced the replication efficiency depending on their lengths. By introducing linker substitutions, we found that the distinct segment required for the efficient replication consisted of an 18-bp sequence, named REE1 (Replication Enhancing Element 1). Single or tandem copies of REE1 alone had no significant replication activity, but they stimulated replication of human chromosomal DNA fragments. We found, in addition, that the REE1 sequence inserted at a site 2.7 kb upstream of the SV40 early promoter caused repression of transcription from the promoter, suggesting that REE1 had a transcriptional silencer activity. Introduction of linker substitutions into the REE1 indicated that the nucleotide sequences required for the repression of transcription were the same as those for enhancement of replication. Thus, REE1 is responsible for both enhancement of replication and repression of transcription.

Autonomous replication of human chromosomal DNA fragments in human cells.

We have examined whether a human chromosome has distinct segments that can replicate autonomously as extrachromosomal elements. Human 293S cells were transfected with a set of human chromosomal DNA fragments of 8-15 kilobase pairs that were cloned on an Escherichia coli plasmid vector. The transfected cells were subsequently cultured in the presence of 5-bromodeoxyuridine during two cell generations, and several plasmid clones labeled in both of the daughter DNA strands were isolated. Efficiency of replication of these clones, as determined from the ratios of heavy-heavy and one-half of heavy-light molecules to total molecules recovered from density-labeled cells, was 9.4% per cell generation on the average. Replication efficiency of control clones excluded during the selection was about 2.2% and that of the vector plasmid alone was 0.3%. A representative clone p1W1 replicated in a semiconservative manner only one round during the S phase of the cell cycle. It replicated extrachromosomally without integration into chromosome. The human segment of the clone was composed of several subsegments that promoted autonomous replication at different efficiencies. Our results suggest that certain specific nucleotide sequences are involved in autonomous replication of human segments.

A mechanism of formation of a persistent hybrid between elongating RNA and template DNA.

At the replication origin of ColE1 plasmid, a persistent hybrid is formed between the primer precursor (RNA II) and its template DNA. The wild-type sequence in the region 13 to 20 bp upstream (-20 region) of the origin is required to form this persistent hybrid. While the template strand for transcription of this region, containing a stretch of six dC residues, is needed, the nontemplate strand can be deleted. Certain mutations in far upstream regions that prevent hybrid formation are suppressed by the nontemplate strand deletion. In RNA II that is forming a persistent hybrid, the region about 265 nucleotides upstream of the origin (-265 region) can also form a hybrid with the template DNA. The -265 region of RNA II that consists of a stretch of six rG residues probably interacts with the dC stretch of the -20 region in the template strand to promote hybrid formation.

A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite.

We report the interaction between a human centromere antigen and an alphoid DNA, a human centromeric satellite DNA, which consists of 170-bp repeating units. A cloned alphoid DNA fragment incubated with a HeLa cell nuclear extract is selectively immunoprecipitated by the anticentromere sera from scleroderma patients. Immunoprecipitation of the DNA made by primer extension defines the 17-bp segment on the alphoid DNA that is required for formation of DNA-antigen complex. On the other hand, when proteins bound to the biotinylated alphoid DNA carrying the 17-bp motif are recovered by streptavidin agarose and immunoblotted, the 80-kD centromere antigen (CENP-B) is detected. DNA binding experiments for proteins immunoprecipitated with anticentromere serum, separated by gel electrophoresis, and transferred to a membrane strongly suggest that the 80-kD antigen specifically binds to the DNA fragment with the 17-bp motif. The 17-bp motif is termed the "CENP-B box." Alphoid monomers with the CENP-B box are found in all the known alphoid subclasses, with varying frequencies, except the one derived from the Y chromosome so far cloned. These results imply that the interaction of the 80-kD centromere antigen with the CENP-B box in the alphoid repeats may play some crucial role in the formation of specified structure and/or function of human centromere.

Transcriptional regulation of early functions of bacteriophage phi 80.

To study the expression of early functions of phi 80 phage, various segments from the early region were transcribed with RNA polymerase. Two major transcripts (from promoters PL and PR) whose synthesis was inhibited by the CI protein were identified. Synthesis of the third major transcript (from promoter PRM) was induced by the CI protein. These studies define two operator-promoter regions, OLPL and ORPRPRM. This mode of transcription from the early region is similar to that of phage lambda. However, there are the following major differences. One is the presence of a p-independent terminator of transcription from promoter PL located immediately after gene N and the absence of a p-dependent terminator that corresponds to tR1 of lambda. The other is the uniqueness of the structure and function of the operators. Both OL and OR operator regions consist of three sites, each containing a highly homologous 19 base-pair sequence. In each site, consensus octanucleotide sequences (half-sites) exhibit dyad symmetry, except in one of the sites where the sequences are arranged tandemly. In addition, each operator region also contains a single half-site. The modes of binding of the CI protein and gene 30 protein to these operator sites are quite different from those of the lambda proteins to the lambda operators. For example, binding of the phi 80 CI protein to the OR1 site is less tight than its binding to the OR2 or OR3 site. The gene 30 protein binds to the OR1 site as tightly as to the OR3 site.

Multiple mechanisms for initiation of ColE1 DNA replication: DNA synthesis in the presence and absence of ribonuclease H.

A transcript (RNA II) of plasmid ColE1 that hybridizes with the template DNA is cleaved by RNAase H and used as a primer by DNA polymerase I. However, the plasmid can replicate in bacteria lacking both enzymes, apparently using a different mechanism of initiation of replication. Here we report in vivo and in vitro studies on initiation of DNA replication in the presence or absence of either or both enzymes. Hybridization of RNA II with the template DNA is always required for initiation. Hybridized RNA II is cleaved by RNAase H to form a primer or used as a primer without cleavage by RNAase H. Hybridization also creates a single-stranded region on the nontranscribed strand that can serve as a template for synthesis of the lagging strand in a reaction that does not require DNA polymerase I. Lagging strand synthesis terminates 17 nucleotides upstream of the normal replication origin, forcing unidirectional replication.

Transcriptional activation of ColE1 DNA synthesis by displacement of the nontranscribed strand.

Plasmid ColE1 can replicate using RNAase H and DNA polymerase I. However, it can also replicate in the absence of these enzymes. In this case, formation of a persistent hybrid between a transcript (RNA II) and the DNA indirectly activates subsequent DNA synthesis, instead of providing a primer as it does in the presence of these enzymes. To activate DNA synthesis, a certain length is required for the hybridized region and the region of minimum length cannot include a palindrome. These results show that the single-stranded region of DNA displaced by the hybridization is responsible for the activation. A single-stranded region was identified on the nontranscribed strand by its enhanced reactivity to dimethyl sulfate. The necessary length for the single-stranded region is at least 40 nucleotides. The region probably provides a site for initial binding of a helicase that further unwinds the template DNA for initiation of DNA synthesis.

Factor-independent termination of transcription in a stretch of deoxyadenosine residues in the template DNA.

The primer transcript of plasmid ColE1 extends beyond the replication origin in either of two different modes. It does or does not form a hybrid with the template DNA. When a stretch of 20 deoxyadenosine residues is inserted into the template strand downstream of the origin, more than 90% of hybridized transcripts and about 10% of unhybridized transcripts terminate at the insert. When the number of inserted residues is reduced to ten, the corresponding values are decreased considerably, while the sites of termination are almost unchanged. A palindrome immediately before the stretch increased the efficiency of termination of unhybridized transcripts. Upon termination of hybridized transcripts, RNA polymerase detaches from the template. A structure made by a hybrid or by folded RNA may affect a property of transcription, and a stretch of deoxyadenosine residues including those beyond the actual site of termination may facilitate detachment of RNA polymerase from the template DNA.

Control of primer formation for ColE1 plasmid replication: conformational change of the primer transcript.

The ColE1 primer transcript (RNA II) hybridizes to the template DNA near the replication origin. The hybridized RNA II is cleaved by RNAase H to form the primer. Hybridization is inhibited by binding of another RNA, RNA I, to RNA II. This binding alters the secondary structure of RNA II even in the region far downstream of the segment where RNA I binds. Similar functional and structural alterations of RNA II are produced by a single base-change within this segment. However, a region of about 200 nucleotides proximal to the 5' end of RNA II that includes the segment can be deleted with little effect on primer formation. By forming an alternative structure, the dispensable region affects folding of a downstream region, and thus participates in regulation of primer formation.

Effects of point mutations on formation and structure of the RNA primer for ColE1 DNA replication.

We report mutations affecting several steps of CoIE1 primer formation. Primer precursor forms the hybrid with the template DNA and is cleaved by RNAase H to form the primer. Point mutations at positions -264, -265, -268, and -308 (base pairs upstream of the replication origin) reduce efficiency of hybrid formation. A suppressor mutation at -18 increases the efficiency for a mutant transcript and reduces that for a wild-type transcript. Therefore, hybrid formation probably starts after transcription passes this position. A mutation at -10 affects the site of cleavage by RNAase H. The site appears to be determined by the distance from a stem-loop structure immediately upstream. A double mutation at -186 and -188 results in formation of an RNA secondary structure that prevents use of an RNAase H-cleaved transcript as primer.

Biologically active recombinant formed through DNA pairing by purified recA protein in vitro.

We have detected in vitro homologous recombination mediated by purified recA protein of Escherichia coli as a recombinant phage produced by using the DNA packaging system of phage lambda. When double-stranded DNA of phage lambda carrying amber mutations is incubated with double-stranded DNA carrying the wild-type genes in the presence of recA protein, Mg++ and ATP, and the DNA packaged, amber+ recombinant phage is produced at a high frequency. This reaction depends completely upon the function of the wild-type recA protein. After incubation of 32P-labeled linear DNA (Form III) with bromouracil-labeled circular DNA (Form I-Form II mixture) in the presence of recA protein, Mg++ and ATP, about 10% of the 32P-counts band at an intermediate density in CsCl equilibrium gradient. This fraction yields a high percentage of the recombinant phage after DNA packaging and shows the alpha-shaped and sigma-shaped joint molecules of linear and circular DNA under the electron microscope. Furthermore, we demonstrate that a non-homologous region inhibits the recombination reaction when it is between the marker concerned and the closer cos end. Our results indicate that recA protein acts directly in the initial step of recombination to join the homologous double-stranded DNA and that the resulting molecule can be matured into the recombinant DNA.

A mutation, bsu, that suppresses temperature-sensitive dnaB function in Escherichia coli K12.

A mutation of Escherichia coli K12 that suppresses certain temperature-sensitive dnaB mutations was identified. The suppressor, named bsu maps very near the dnaG mutations. The bsu mutation in dnaB bacteria appears to be dominant over the wild-type allele, and suppresses specifically the temperature-sensitive dnaB mutations which are revertible phenotypically when salt is present. The observed specificity in suppression suggests that the product of bsu alone cannot substitute for the defective dnaB gene products. These findings suggest strongly that gene products of bsu and dnaB interact with each other in the process of DNA replication in E. coli.