Eukaryotic DNA replication: from pre-replication complex to initiation complex

A common mechanism has emerged for the control of the initiation of eukaryotic DNA replication. The minichromosome maintenance protein complex (MCM) and Cdc45 have now been recognized as central components of the initiation machinery. In addition, two types of S phase promoting kinases conserved between yeast and humans play critical roles in the initiation reaction. At the onset of S phase, S phase kinases promote the association of Cdc45 with MCM at origins. Upon the formation of the MCM-Cdc45 complex at origins, the duplex DNA is unwound and various replication proteins, including DNA polymerases, are recruited onto unwound DNA. The increasing number of newly identified factors involved in the initiation reaction indicates that the control of initiation requires highly evolved machinery in eukaryotic cells.     

DNMT1 is a component of a multiprotein DNA replication complex

DNA methylation is a major determinant of epigenetic inheritance and plays an important role in genome stability. The accurate propagation of DNA methylation patterns with cell division requires that methylation be closely coupled to DNA replication, however the precise molecular determinants of this interaction have not been defined. In the present study, we show that the predominant DNA methyltransferase species in somatic cells, DNMT1, is a component of a multiprotein DNA replication complex termed the DNA synthesome that fully supports semi-conservative DNA replication in a cell-free system. DNMT1 protein and activity were found to co-purify with the human DNA synthesome through a series of subcellular fractionation and chromatography steps, resulting in an enrichment of methyltransferase specific activity from two human cell lines. DNA methyltransferase activity co-eluted with in vitro replication activity and DNA polymerase alpha activity on sucrose density gradients suggesting that DNMT1 is a tightly bound, core component of the replication complex. The synthesome-associated pool of DNA methyltransferase exhibited both maintenance and de novo methyltransferase activity and the ratio of the two was similar to that observed in whole cell lysates and for recombinant DNMT1. These data indicate that interactions within the synthesome complex do not influence the intrinsic preference of DNMT1 for hemimethylated DNA, but suggest that newly replicated DNA may be subject to low level de novo methylation. The data indicate that DNA methylation is tightly coupled to replication through physical interaction of DNMT1 and core components of the replication machinery. The definition of the molecular interactions between DNMT1 and other proteins in the replication complex in normal and neoplastic cells will provide further insight into the regulation of DNA methylation and the mechanisms underlying the alteration of DNA methylation patterns during carcinogenesis.     

The clamp-loading complex for processive DNA replication

DNA polymerase requires two processing factors, sliding clamps and clamp loaders, to direct rapid and accurate duplication of genomic DNA. In eukaryotes, proliferating cell nuclear antigen (PCNA), the ring-shaped sliding clamp, encircles double-stranded DNA within its central hole and tethers the DNA polymerases onto DNA. Replication factor C (RFC) acts as the clamp loader, which correctly installs the sliding clamp onto DNA strands in an ATP-dependent manner. Here we report the three-dimensional structure of an archaeal clamp-loading complex (RFC-PCNA-DNA) determined by single-particle EM. The three-dimensional structure of the complex, reconstituted in vitro using a nonhydrolyzable ATP analog, reveals two components, a closed ring and a horseshoe-shaped element, which correspond to PCNA and RFC, respectively. The atomic structure of PCNA fits well into the closed ring, suggesting that this ternary complex represents a state just after the PCNA ring has closed to encircle the DNA duplex.     

Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex.

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.     

DNA replication: making two forks from one prereplication complex

The copying of chromosomal DNA initiates from a single nucleoprotein assembly called the prereplication complex. New findings in a recent issue of Molecular Cell (Yardimci et?al., 2010) reveal that this complex dissolves into two independent replisomes that move away from each other as DNA synthesis ensues.     

Human cell DNA replication is mediated by a discrete multiprotein complex

A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell.     

Cdc7 kinase complex: a key regulator in the initiation of DNA replication

DNA replication results from the action of a staged set of highly regulated processes. Among the stages of DNA replication, initiation is the key point at which all the G1 regulatory signals culminate. Cdc7 kinase is the critical regulator for the ultimate firing of the origins of initiation. Cdc7, originally identified in budding yeast and later in higher eukaryotes, forms a complex with a Dbf4-related regulatory subunit to generate an active kinase. Genetic evidence in mammals demonstrates essential roles for Cdc7 in mammalian DNA replication. Mini-chromosome maintenance protein (MCM) is the major physiological target of Cdc7. Genetic studies in yeasts indicate additional roles of Cdc7 in meiosis, checkpoint responses, maintenance of chromosome structures, and repair. The interplay between Cdc7 and Cdk, another kinase essential for the S phase, is also discussed.     

The complex of DNA gyrase and quinolone drugs on DNA forms a barrier to the T7 DNA polymerase replication complex

Quinolone drugs can inhibit bacterial DNA replication, via interaction with the type II topoisomerase DNA gyrase. Using a DNA template containing a preferred site for quinolone-induced gyrase cleavage, we have demonstrated that the passage of the bacteriophage T7 replication complex is blocked in vitro by the formation of a gyrase-drug-DNA complex. The majority of the polymerase is arrested approximately 10 bp upstream of this preferred site, although other minor sites of blocking have been observed. The ability of mutant gyrase proteins to arrest DNA replication in vitro has been investigated. Gyrase containing mutations in the A subunit at either the active-site tyrosine (Tyr122) or Ser83 (a residue known to be involved in quinolone interaction) failed to halt the progress of the polymerase. A low-level, quinolone-resistant mutation in the B subunit of gyrase showed reduced blocking compared to wild-type. We have demonstrated that DNA cleavage and replication blocking occur on similar time-scales and we conclude that formation of the cleavable complex is a prerequisite for polymerase blocking. Additionally, we have shown that collision of the replication proteins with the gyrase-drug-DNA complex is not sufficient to render this complex irreversible and that further factors must be involved in processing this stalled complex.     

Rescuing stalled replication forks: MMS22L-TONSL,a novel complex for DNA replication fork repair in human cells

Comment on: Piwko W, et al. EMBO J 2010; 29:4210-22, Duro E, et al. Mol Cell 2010; 40:632–44, O’Connell BC, et al. Mol Cell 2010; 40:645–57 and O’Donnell L, et al. Mol Cell 2010; 40:619–31.     

Directionality of lambda plasmid DNA replication carried out by the heritable replication complex

There are two ‘pathways’ of replication of λ plasmids in Escherichia coli. One pathway requires the assembly of a new replication complex before replication and the second pathway is based on the activity of the replication complex inherited by one of two daughter plasmid copies after a preceding replication round. Such a phenomenon was postulated to occur also in other replicons, including Saccharomyces cerevisiae autonomously replicating sequences. Here we investigated directionality of λ plasmid replication carried out by the heritable and newly assembled replication complexes. Using two-dimensional agarose gel electrophoresis and electron microscopy we demonstrated that in both normal growth conditions and during the relaxed response to amino acid starvation (when only replication carried out by the heritable complex is possible), bidirectionally and undirectionally replicating plasmid molecules occurred in host cells in roughly equal proportions. The results are compatible with the hypothesis that both complexes (heritable and newly assembled) are equivalent.     

Direct interaction between cohesin complex and DNA replication machinery

Structural maintenance of chromosome 1 (Smc1) is a multifunctional protein, which has been implicated in sister chromatid cohesion, DNA recombination and repair, and the activation of cell cycle checkpoints by ionizing radiation, ultraviolet light, and other genotoxic agents. In order to identify the proteins that interact with Smc1, we conducted the Tandem affinity purification (TAP) technique and analyzed the Smc1-interacting proteins via MALDI-TOF mass spectrometry. We identified minichromosome maintenance 7 (Mcm7), an essential component of the pre-replication complex, as a novel Smc1-interacting protein. Co-immunoprecipitation revealed an interaction occurring between Smc1 and Mcm7, both in vitro and in vivo. Using a GST pull-down assay, we determined that Smc1 interacts physically with Mcm7 via its N-terminal and hinge regions, and Mcm7 interacts with Smc1 via its middle region. Interestingly, we also discovered that Smc1 interacts with other DNA replication proteins, including Mcm6, RFC1, and DNA polymerase alpha. These results suggest that a functional link exists between the cohesin complex and DNA replication proteins.     

Evidence for participation of a multiprotein complex in yeast DNA replication in vitro

Fractions containing a high molecular weight form (Mr approximately equal to 2 X 10(6] of the activity that replicates in vitro both the 2-micron yeast DNA plasmid and the chromosomal autonomously replicating sequence ars 1 can be prepared from cells of the budding yeast Saccharomyces. Protein complexes from the fractions associate in vitro with the replication origins of these DNA elements, as determined by electron microscopy. In the present study, the high molecular weight replicative fraction has been characterized in further detail. The DNA synthetic activity in the high molecular weight fraction was bound to the DNA and could be isolated with it. This binding of the replicating activity to the DNA was greatly reduced in the absence of the 2-micron origins of replication. Association of the protein complexes with DNA depended on the amount of replicating activity added, was sensitive to 0.2 M KCl, and exhibited a requirement for rATP and deoxyribonucleoside triphosphates. It was not blocked, however, by the DNA polymerase inhibitor aphidicolin or by the RNA polymerase inhibitor alpha-amanitin. The lack of inhibition by aphidicolin suggests that the deoxyribonucleoside triphosphates may function as cofactors in the binding of protein complexes to DNA or as substrates for a polymerizing activity such as a primase. Binding of the protein complexes as well as actual DNA replication were heat sensitive in the high molecular weight fraction prepared from the temperature-sensitive mutant of the cell division cycle cdc 8. This suggests that the cdc 8 gene product is present in a replicative protein complex and strengthens the conclusion that the presence of the protein complexes on the DNA is associated with replication. Using independent enzyme assays, several other possible replication proteins (including DNA polymerase I, DNA ligase, DNA primase, and DNA topoisomerase II) have been identified directly in the high molecular weight replicative fraction. All of these results provide support for the idea that a protein complex (or replisome ) is involved in the replication of both the extrachromosomal 2-micron DNA and chromosomal DNA in yeast.     

A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis

Two dimers of the replication terminator protein (RTP) of Bacillus subtilis bind to a chromosomal DNA terminator site to effect polar replication fork arrest. Cooperative binding of the dimers to overlapping half-sites within the terminator is essential for arrest. It was suggested previously that polarity of fork arrest is the result of the RTP dimer at the blocking (proximal) side within the complex binding very tightly and the permissive-side RTP dimer binding relatively weakly. In order to investigate this "differential binding affinity" model, we have constructed a series of mutant terminators that contain half-sites of widely different RTP binding affinities in various combinations. Although there appeared to be a correlation between binding affinity at the proximal half-site and fork arrest efficiency in vivo for some terminators, several deviated significantly from this correlation. Some terminators exhibited greatly reduced binding cooperativity (and therefore have reduced affinity at each half-site) but were highly efficient in fork arrest, whereas one terminator had normal affinity over the proximal half-site, yet had low fork arrest efficiency. The results show clearly that there is no direct correlation between the RTP binding affinity (either within the full complex or at the proximal half-site within the full complex) and the efficiency of replication fork arrest in vivo. Thus, the differential binding affinity over the proximal and distal half-sites cannot be solely responsible for functional polarity of fork arrest. Furthermore, efficient fork arrest relies on features in addition to the tight binding of RTP to terminator DNA.