The human homologue of fission Yeast cdc27, p66, is a component of active human DNA polymerase delta.

An essential eukaryotic DNA polymerase, DNA polymerase delta (pol delta), synthesizes DNA processively in the presence of proliferating cell nuclear antigen (PCNA). Recently, a 66 kDa polypeptide (p66) that displays significant homology within its PCNA binding domain to that of fission yeast cdc27 was identified as a component of mouse and calf thymus pol delta. Our studies show that p66 interacts tightly with other subunits of pol delta during size fractionation of human cell extracts, and co-immunoprecipitates with these subunits along with PCNA-dependent polymerase activity. Active human pol delta could be reconstituted by co-expressing p125, p50, and p66 recombinant baculoviruses, but not by co-expressing p125 and p50 alone. Interaction studies demonstrated that p66 stabilizes the association between p125 and p50. Pull-down assays with PCNA-linked beads demonstrated that p66 increases the overall affinity of pol delta for PCNA. These results indicate that p66 is a functionally important subunit of human pol delta that stabilizes the pol delta complex and increases the affinity of pol delta for PCNA.

ATP-dependent structural change of the eukaryotic clamp-loader protein, replication factor C.

The eukaryotic DNA sliding clamp that keeps DNA polymerase engaged at a replication fork, called proliferating cell nuclear antigen (PCNA), is loaded onto the 3' ends of primer DNA through its interaction with a heteropentameric protein complex called replication factor C (RFC). The ATPase activity of RFC is necessary for formation of a functional PCNA clamp. In the present study, the sensitivity of RFC to partial proteolysis is used to show that addition of ATP, ATPgammaS, or ADP induces different structural changes in RFC. Direct observation by electron microscopy reveals that RFC has a closed two-finger structure called the U form in the absence of ATP. This is converted into a more open C form on addition of ATP. In contrast, the structural changes induced by ATPgammaS or ADP are limited. These results suggest that RFC adapts on opened configuration intermediately after ATP hydrolysis. We further observe that PCNA is held between the two fingers of RFC and propose that the RFC structure change we observe during ATP hydrolysis causes the attached PCNA to form its active ring-like clamp on DNA.

Association of human origin recognition complex 1 with chromatin DNA and nuclease-resistant nuclear structures.

An origin recognition complex (ORC) consisting of six polypeptides has been identified as a DNA replication origin-binding factor in Saccharomyces cerevisiae. Homologues of ORC subunits have been discovered among eukaryotes, and we have prepared monoclonal antibodies against a human homologue of ORC1 (hORC1) to study its localization in human cells. It was thus found to associate with nuclei throughout the cell cycle and to be resistant to nonionic detergent treatment, in contrast to MCM proteins, which are other replication factors, the association of which with nuclei is clearly dependent on the phase of the cell cycle. A characteristic feature of hORC1 is dissociation by NaCl in a narrow concentration range around 0.25 M, suggesting interaction with some specific partner(s) in nuclei. Nuclease treatment experiments and UV cross-linking experiments further indicated interaction with both nuclease-resistant nuclear structures and chromatin DNA. Although its DNA binding was unaffected, some variation in the cell cycle was apparent, the association with nuclear structures being less stable in the M phase. Interestingly, the less stable association occurred concomitantly with hyperphosphorylation of hORC1, suggesting that this hyperphosphorylation may be involved in M phase changes.

Regulation of DNA-replication origins during cell-cycle progression.

We have shown previously that chromosome VI of Saccharomyces cerevisiae contains nine origins of DNA replication that differ in initiation frequency and replicate sequentially during the S phase of the cell cycle. Here we show that there are links between activation of these multiple origins and regulation of S-phase progression. We study the effects of a DNA-damaging agent, methyl methane sulphonate (MMS), and of mutations in checkpoint genes such as rad53 on the activity of origins, measured by two-dimensional gel analysis, and on cell-cycle progression, measured by fluorescence-activated cell sorting. We find that when MMS slows down S-phase progression it also selectively blocks initiation from late origins. A rad53 mutation enhances late and/or inefficient origins and releases the initiation block by MMS. Mutation of rad53 also results in a late origin becoming early replicating. We conclude that rad53 regulates the timing of initiation of replication from late origins during normal cell growth and blocks initiation from late origins in MMS-treated cells. rad53 is, therefore, involved in the cell's surveillance of S-phase progression. We also find that orc2, which encodes subunit 2 of the origin-recognition complex, is involved in suppression of late origins.

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.

Cell cycle dependent topological changes of chromosomal replication origins in Saccharomyces cerevisiae.

BACKGROUND: The ORC (Origin Recognition Complex) of Saccharomyces cerevisiae is a protein complex for the initiation of replication which interacts with a cis-element, ACS (ARS Consensus Sequence), essential for DNA replication. The protein-DNA complex detected by the DNase I genomic footprinting method has been shown to vary depending on cell cycle progression. Further studies on topological changes of replication origin in vivo caused by ORC association are crucial for an understanding of chromosomal DNA replication in S. cerevisiae. RESULTS: Topological changes in the replication origins of the S. cerevisiae chromosome were studied by an in vivo UV photofootprinting method which is capable of detecting the change in the flexibility of DNA caused by protein binding. The footprinting method detected the inhibition and enhancement of UV-induced pyrimidine dimer formation in A and B1 elements of a chromosomal origin, ARS1, depending on the activity of native ORC subunits. Furthermore, footprint patterns were reproduced in vitro with purified ORC. The inhibition regarding the A element was stronger during the S to late M phase than that during the progression through the G1 phase. Functional CDC6 and MCM5 were required for maintaining the weaker inhibition state in G1-arrested cells. CONCLUSION: The application of in vivo UV photofootprinting in studies of topological changes of S. cerevisiae replication origins revealed the presence of two modes of topological ORC-ACS interaction. The weaker footprint in the G1 phase represents a specific topology of ACS, resulting from an alteration of the ORC-ACS interaction aided by CDC6 and MCM5, and this topological change may make the replication origin competent for initiating DNA replication.

The efficiency and timing of initiation of replication of multiple replicons of Saccharomyces cerevisiae chromosome VI.

BACKGROUND: A complete set of nine ARSs was identified (the tenth ARS in this paper), mapped on chromosome VI of Saccharomyces cerevisiae, and characterized for functional elements. RESULTS: The level of activity of all ARSs as chromosomal replication origins was determined by neutral/neutral 2D gel-electrophoresis. These origins were classified into three groups: (i) three high frequency origins used once nearly every cell cycle, (ii) four intermediate frequency origins used once in two to three cycles and (iii) two low frequency origins used in fewer than 5% of cell cycles. These variations in initiation frequency among origins of chromosome VI are present in three common laboratory wild-type strains. Each origin is initiated at a fixed time and passively replicated by incoming replication forks at a fixed time during a synchronous S phase. Replication of each arm of the chromosome starts from one major origin located one-fifth (left arm) and one-third (right arm) of the distance from the centromere, and expands sequentially in both directions. Two telomere vicinity origins are replicated last. Time of initiation and replication of the last replicating origin, Ori609, was remarkably variable from cell to cell. CONCLUSIONS: Chromosome VI of S. cerevisiae contains nine replication origins that comprise five active replicons under normal cell growth conditions. A clear correlation was found between the efficiency of initiation and the order of replication. The timing of initiation of most origins, except for the first and last, is coincident with the time of passive replication by incoming forks from neighbouring origins.

Characterization of a novel CDC gene (ORC1) partly homologous to CDC6 of Saccharomyces cerevisiae.

A novel cell cycle gene was identified by a computer search for genes partly homologous to known CDC genes, CDC6 of Saccharomyces cerevisiae and CDC18 of Schizosaccharomyces pombe, using the nucleotide sequence data base for S. cerevisiae produced by the Yeast Sequencing Project. The protein sequence coded by the cloned gene was found to be identical to that of purified ORC1 protein. Disruption of the gene and subsequent tetrad analysis revealed that the gene was essential for growth. The function of the gene product was analyzed by depleting the protein from the cell using a mutant haploid strain containing the disrupted ORC1 gene on the chromosome and a galactose-inducible gene coding for HA-tagged ORC1 protein on a single copy plasmid. The HA-tagged protein was expressed during growth in the presence of galactose but began to decrease rapidly upon depletion of galactose. Analysis of the cell cycle progression of the mutant cells by FACS after the removal of galactose from the medium, and microscope observations of cells and their nuclei revealed that the normal progression of 2N cells was immediately impeded as the ORC1 protein started to decrease. This was blocked completely in the cells that had progressed to the S phase under conditions deficient in ORC1 protein followed by cell death. Two-dimensional gel analysis of the replication intermediates after the galactose removal revealed that the depletion of ORC1 protein caused a decrease in the frequency of initiation of chromosomal replication, eventually resulting in the inhibition of replication as a whole. The function of the ORC1 protein in the cell cycle progression of S. cerevisiae is discussed in light of current information on ORC.

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.