Deoxynucleoside triphosphate (dNTP) synthesis and destruction regulate the replication of both cell and virus genomes

Biochemical reactions, even those as complex as replicating the DNA genome of cells, follow the principle that the process is regulated by both the substrate concentration and by the enzymes that mediate the process. Deoxynucleoside triphosphates (dNTPs), the substrates for DNA polymerizing enzymes, have long been known to be limited in their concentration in cells because the enzyme that synthesizes deoxynucleotides from ribonucleotides, ribonucleotide reductase (RNR), is synthesized and enzymatically activated as cells enter the S phase (1, 2). RNR, discovered by Peter Reichard 52 y ago (3), converts all four ribonucleotide diphosphates (rNDPs) to the respective deoxynucleoside disposphates (dNDPs), which are then rapidly converted to dNTP. Low levels and activity of RNR provide sufficient dNTPs for mitochondrial DNA synthesis and for DNA repair in noncycling cells and during the G1 phase of the cell-division cycle in proliferating cells, but RNR levels and activity are hugely increased as cells commit to replicate DNA during the S phase of the cell-division cycle or following extensive DNA repair (4). Indeed, RNR is one of the most highly regulated enzymes known. The mammalian enzyme synthesizes all four dNDPs in a cycle, is allosterically activated by dATP, dTTP, and dGTP to balance the relative levels of the four dNTPs (dCTP, dTTP, dGTP and dATP), and is feed-back–inhibited by dATP, because dATP is the last dNTP to be made in the cycle of synthesizing all four dNTPs by a single RNR enzyme (1). Specific inhibitory proteins (in yeasts) also control RNR activity and RNR subunit levels are regulated by cell cycle-dependent transcription of the genes encoding the subunits and by subunit protein stability (4, 5). On the basis of these observations, one might expect that dNTP synthesis by RNR should be sufficient to control how and when genome DNA replication occurs because RNR is only maximally active during the S phase. However, recent studies, including those emerging from far-afield studies of how HIV replication is restricted to certain cell types (6, 7), have uncovered a new control of dNTP levels, dNTP destruction. The sterile alpha motif and HD-domain containing protein 1 (SAMHD1) protein is a deoxynucleoside triphosphohydrolase that cleaves dNTPs to the respective deoxynucleoside and a triphosphate (8). In PNAS, Franzolin et al. (9) show that dNTP destruction by SAMHD1 also contributes to dNTP concentration control during the cell-division cycle of proliferating cells, thereby affecting both DNA replication and cell-cycle progression

ATP dependent assembly of the human origin recognition complex

The Origin Recognition Complex (ORC) was initially discovered in budding yeast extracts as a protein complex that binds with high affinity to Autonomously Replicating Sequences (ARS) in an ATP dependent manner. We have cloned and expressed the human homologs of the ORC subunits as recombinant proteins. In contrast to other eukaryotic initiators examined thus far, assembly of human ORC in vitro is dependent on ATP binding. Mutations in the ATP binding sites of Orc4 or Orc5 impair complex assembly, whereas Orc1 ATP binding is not required. Immunofluorescence staining of human cells with anti-Orc3 antibodies demonstrate cell cycle-dependent association with a nuclear structure. Immunoprecipitation experiments show that ORC disassembles as cells progress through S phase. The Orc6 protein binds directly to the Orc3 subunit and interacts as part of ORC in vivo. These data suggest that the assembly and disassembly of ORC in human cells is uniquely regulated and may contribute to restricting DNA replication to once in every cell division cycle

Cdc6 ATPase activity regulates ORC center dot Cdc6 stability and the selection of specific DNA sequences as origins of DNA replication

DNA replication, as with all macromolecular synthesis steps, is controlled in part at the level of initiation. Although the origin recognition complex ( ORC) binds to origins of DNA replication, it does not solely determine their location. To initiate DNA replication ORC requires Cdc6 to target initiation to specific DNA sequences in chromosomes and with Cdt1 loads the ring-shaped mini-chromosome maintenance ( MCM) 2-7 DNA helicase component onto DNA. ORC and Cdc6 combine to form a ring-shaped complex that contains six AAA(+) subunits. ORC and Cdc6 ATPase mutants are defective in MCM loading, and ORC ATPase mutants have reduced activity in ORC.Cdc6.DNA complex formation. Here we analyzed the role of the Cdc6 ATPase on ORC.Cdc6 complex stability in the presence or absence of specific DNA sequences. Cdc6 ATPase is activated by ORC, regulates ORC.Cdc6 complex stability, and is suppressed by origin DNA. Mutations in the conserved origin A element, and to a lesser extent mutations in the B1 and B2 elements, induce Cdc6 ATPase activity and prevent stable ORC.Cdc6 formation. By analyzing ORC.Cdc6 complex stability on various DNAs, we demonstrated that specific DNA sequences control the rate of Cdc6 ATPase, which in turn controls the rate of Cdc6 dissociation from the ORC.Cdc6.DNA complex. We propose a mechanism explaining how Cdc6 ATPase activity promotes origin DNA sequence specificity; on DNA that lacks origin activity, Cdc6 ATPase promotes dissociation of Cdc6, whereas origin DNA down-regulates Cdc6 ATPase resulting in a stable ORC.Cdc6.DNA complex, which can then promote MCM loading. This model has relevance for origin specificity in higher eukaryotes

An interaction between replication protein A and SV40 T antigen appears essential for primosome assembly during SV40 DNA replication

Replication protein A from human cells (hRPA) is a multisubunit single-stranded DNA-binding protein (ssb) and is essential for SV40 DNA replication in vitro. The related RPA from Saccharomyces cerevisiae (scRPA) is unable to substitute for hRPA in SV40 DNA replication. To understand this species specificity, we evaluated human and yeast RPA in enzymatic assays with SV40 T antigen (TAg) and human DNA polymerase alpha/primase, the factors essential for initiation of SV40 DNA replication. Both human and yeast RPA stimulated the polymerase and (at subsaturating levels of RPA) the primase activities of human DNA polymerase alpha/primase on homopolymer DNA templates. In contrast, both human and yeast RPA inhibited synthesis by DNA polymerase alpha/primase on natural single-stranded DNA (ssDNA) templates. T antigen reversed the inhibition of DNA polymerase alpha/primase activity on hRPA-coated natural ssDNA, as previously described, but was unable to reverse the inhibition on scRPA or Escherichia coli ssb-coated templates. Therefore, the ability of an ssb to reconstitute SV40 DNA replication correlated with its ability to allow the TAg stimulation of polymerase alpha/primase in this assay. Enzyme-linked immunoassays demonstrated that hRPA interacts with TAg, as previously described; however, scRPA does not bind to TAg in this assay. These and other recent results suggest that T antigen contains a function analogous to some prokaryotic DNA replication proteins that facilitate primosome assembly on ssb-coated template DNAs

DNA binding properties of an HMG1-related protein from yeast mitochondria

The DNA binding properties of ABF2, an abundant protein found in the mitochondria of the yeast Saccharomyces cerevisiae have been examined in detail. ABF2 is closely related to the vertebrate high mobility group protein HMG1 and like HMG1, ABF2 will introduce negative supercoils into a relaxed, double-stranded circular DNA molecule in cooperation with a DNA topoisomerase. Additionally, ABF2 binds approximately 5-10 times more tightly to negatively supercoiled DNA than to relaxed circular or linear DNA. Although ABF2 binds to most random double-stranded sequences with roughly equal affinity, its binding within certain key regulatory regions is qualitatively quite different. First, ABF2 binding induces a distinct pattern of DNA bending within the chromosomal origin of DNA replication, ARS1. Second, ABF2 binding to all nuclear replication origins tested, in addition to a critical mitochondrial promoter and replication origin, is clearly nonrandom as visualized by DNase1 footprinting. Analysis of the sequences found within these regions as well as competition experiments with synthetic DNA molecules suggest that site-specific DNA binding may be accomplished by the phased distribution of short stretches of poly(dA), which exclude ABF2 binding. These patterns of ABF2 DNA binding suggest a role for the protein in genome organization and site-specific regulation of transcription or DNA replication

Replication factor-A from Saccharomyces cerevisiae is encoded by three essential genes coordinately expressed at S phase

Replication factor-A (RF-A) is a three-subunit protein complex originally purified from human cells as an essential component for SV40 DNA replication in vitro. We have previously identified a functionally homologous three-subunit protein complex from the yeast Saccharomyces cerevisiae. Here we report the cloning and characterization of the genes encoding RF-A from S. cerevisiae. Each of the three subunits is encoded by a single essential gene. Cells carrying null mutations in any of the three genes arrest as budded and multiply budded cells. All three genes are expressed in a cell-cycle-dependent manner; the mRNA for each subunit peaks at the G1/S-phase boundary. A comparison of protein sequences indicates that the human p34 subunit is 29% identical to the corresponding RFA2 gene product. However, expression of the human protein fails to rescue the rfa2::TRP1 disruption

Concerted activities of Mcm4, Sld3 and Dbf4 in control of origin activation and DNA replication fork progression

Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins in a temporally specific manner during S phase. The replicative helicase Mcm2-7 functions in both initiation and fork progression and thus is an important target of regulation. Mcm4, a helicase subunit, possesses an unstructured regulatory domain that mediates control from multiple kinase signaling pathways, including the Dbf4-dependent Cdc7 kinase (DDK). Following replication stress in S phase, Dbf4 and Sld3, an initiation factor and essential target of Cyclin-Dependent Kinase (CDK), are targets of the checkpoint kinase Rad53 for inhibition of initiation from origins that have yet to be activated, so-called late origins. Here, whole genome DNA replication profile analysis is employed to access under various conditions the effect of mutations that alter the Mcm4 helicase regulatory domain and the Rad53 targets, Sld3 and Dbf4. Late origin firing occurs under genotoxic stress when the controls on Mcm4, Sld3 and Dbf4 are simultaneously eliminated. The regulatory domain of Mcm4 plays an important role in the timing of late origin firing, both in an unperturbed S phase and dNTP limitation. Furthermore, checkpoint control of Sld3 impacts fork progression under replication stress. This effect is parallel to the role of the Mcm4 regulatory domain in monitoring fork progression. Hypomorph mutations in sld3 are suppressed by a mcm4 regulatory domain mutation. Thus, in response cellular conditions, the functions executed by Sld3, Dbf4 and the regulatory domain of Mcm4 intersect to control origin firing and replication fork progression, thereby ensuring genome stability

Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

Newly-born cells either continue to proliferate or exit the cell division cycle. This decision involves delaying expression of Cyclin E that promotes DNA replication. ORC1, the Origin Recognition Complex (ORC) large subunit, is inherited into newly-born cells after it binds to condensing chromosomes during the preceding mitosis. We demonstrate that ORC1 represses Cyclin E gene (CCNE1) transcription, an E2F1 activated gene that is also repressed by the Retinoblastoma (RB) protein. ORC1 binds to RB, the histone methyltransferase SUV39H1 and to its repressive histone H3K9me3 mark. ORC1 cooperates with SUV39H1 and RB protein to repress E2F1-dependent CCNE1 transcription. In contrast, the ORC1-related replication protein CDC6 binds Cyclin E-CDK2 kinase and in a feedback loop removes RB from ORC1, thereby hyper-activating CCNE1 transcription. The opposing effects of ORC1 and CDC6 in controlling the level of Cyclin E ensures genome stability and a mechanism for linking directly DNA replication and cell division commitment

Immunological characterization of chromatin assembly factor I, a human cell factor required for chromatin assembly during DNA replication in vitro

Chromatin assembly factor I (CAF-I) is a multisubunit protein complex purified from the nuclei of human cells and required for chromatin assembly during DNA replication in vitro. Purified CAF-I promotes chromatin assembly in a reaction that is dependent upon, and coupled with, DNA replication and is therefore likely to reflect events that occur during S phase in vivo. In order to investigate the regulation and mechanism of CAF-I and the replication-dependent chromatin assembly process, we have used the purified protein to raise monoclonal antibodies. In this report we describe the characterization of a panel of monoclonal antibodies which recognize different subunits of the CAF-I complex. We use immunoprecipitation analysis to show that CAF-I exists as a multiprotein complex in vivo and that some of the polypeptides are phosphorylated. In addition, immunocytochemistry demonstrates that CAF-I is localized to the nucleus of human cells. Finally, monoclonal antibodies directed against the individual subunits of CAF-I immunodeplete chromatin assembly activity from nuclear extracts, confirming that CAF-I is a multisubunit protein required for chromatin assembly in vitro