Requirement for the replication protein SSB in human DNA excision repair

Replication and repair are essential processes that maintain the continuity of the genetic material. Dissection of simian virus 40 (SV40) DNA replication has resulted in the identification of many eukaryotic replication proteins, but the biochemistry of the multienzyme process of DNA excision repair is less well defined. One protein that is absolutely required for semiconservative replication of SV40 DNA in vitro is human single-stranded DNA-binding protein (SSB, also called RF-A and RP-A). SSB consists of three polypeptides of relative molecular mass 70,000, 34,000 and 13,000, and acts with T antigen and topoisomerases to unwind DNA, allowing the access of other replication proteins. Human SSB can also stimulate the activity of polymerases alpha and delta, suggesting a further role in elongation during DNA replication. We have now found a role for human SSB in DNA excision repair using a cell-free system that can carry out nucleotide excision repair in vitro. Monoclonal antibodies against human SSB caused extensive inhibition of DNA repair in plasmid molecules damaged by ultraviolet light or acetylaminofluorene. Addition of purified SSB reversed this inhibition and further stimulated repair synthesis by increasing the number of repair events. These results show that a mammalian DNA replication protein is also essential for repair.     

Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein

The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.     

The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro

Cell-free extracts prepared from human 293 cells, supplemented with purified SV40 large-T antigen, support replication of plasmids containing the SV40 origin of DNA replication. A cellular protein (Mr approximately 36,000) that is required for efficient SV40 DNA synthesis in vitro has been purified from these extracts. This protein is recognized by human autoantibodies and is identified as the cell-cycle regulated protein known as proliferating cell nuclear antigen (PCNA) or cyclin.     

ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication

Simian virus 40 (SV40) replicates in nuclei of human and monkey cells. One viral protein, large tumour (T) antigen, is required for the initiation of DNA replication. The development of in vitro replication systems which retain this property has facilitated the identification of the cellular components required for replication. T antigen recognizes the pentanucleotide 5'-GAGGC-3' which is present in four copies within the 64 base-pairs (bp) of the core origin. In the presence of ATP it binds with increased affinity forming a distinctive, bilobed structure visible in electron micrographs. As a helicase, it unwinds SV40 DNA bidirectionally from the origin. We report here that in vitro and in the presence of ATP, T antigen assembles a double hexamer, centred on the core origin and extending beyond it by 12 bp in each direction. The assembly of this dodecamer initiates an untwisting of the duplex by 2-3 turns. In the absence of ATP, a tetrameric structure is the largest found at the core origin. In the absence of DNA, but in the presence of ATP or its non-hydrolysable analogues, T antigen assembles into hexamers. This suggests that ATP effects an allosteric change in the monomer. The change alters protein-protein interactions and allows the assembly of a double hexamer, which initiates replication at the core origin.     

Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo

Evolutionarily diverse eukaryotic cells share many conserved proteins of unknown function. Some are essential for cell viability, emphasising their importance for fundamental processes of cell biology but complicating their analysis. We have developed an approach to the large-scale characterization of such proteins, based on conditional and rapid degradation of the target protein in vivo, so that the immediate consequences of bulk protein depletion can be examined. Budding yeast strains have been constructed in which essential proteins of unknown function have been fused to a 'heat-inducible-degron' cassette that targets the protein for proteolysis at 37 degrees C (ref. 4). By screening the collection for defects in cell-cycle progression, here we identify three DNA replication factors that interact with each other and that have uncharacterized homologues in human cells. We have used the degron strains to show that these proteins are required for the establishment and normal progression of DNA replication forks. The degron collection could also be used to identify other, essential, proteins with roles in many other processes of eukaryotic cell biology.     

Simian virus 40 replication in adenovirus-transformed human cells antagonizes gene expression

Simian virus 40 (SV40) replicates efficiently in monkey kidney cells. However, we have now found that SV40-based vectors transfected into most human cells replicate poorly, if at all. In contrast, strong SV40 replication is observed in human embryonic kidney (HEK) cells transformed with the adenovirus early region, but not in untransformed HEK cells. Vector replication in adenovirus-transformed cells is dependent on the presence of the SV40 origin of replication and large-T antigen. However, vigorous replication occurs at levels of large-T antigen that are undetectable by immunofluorescence. These data suggest that the adenovirus oncogenes create a replication-permissive environment to which the SV40 replicon responds. Furthermore, replication and gene expression seem to be antagonistic on our vectors. High levels of large-T antigen are observed only when vector replication is blocked by mutations in the gene for large-T antigen or the origin of replication, or by direct inhibition of DNA polymerase with aphidicolin.     

XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis

In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref. 1), a protein which allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase.     

Mouse p53 inhibits SV40 origin-dependent DNA replication

p53 is a cellular phosphoprotein that is present at elevated concentrations in cells transformed by different agents. p53 complementary DNA expression-constructs immortalize primary cells in vitro and co-operate with an activated ras oncogene in malignant transformation. Several reports have implicated p53 in mammalian cell cycle control and specifically with events occurring at the G0-G1 boundary. p53 forms specific complexes with simian virus 40 (SV40) large-T antigen, and such complexes are found associated with both replicating and mature SV40 DNA in lytically infected cells. In an accompanying paper Gannon and Lane report that in in vitro plate-binding assays, mouse p53 can displace polymerase alpha from complex with T-antigen. We have examined the in vivo consequences of expressing wild-type and mutant p53 proteins from other species in SV40-transformed monkey cells. We report here that expression of mouse p53 results in a substantial and selective inhibition of SV40 origin-dependent DNA replication. In addition to any function in the G0-G1 transition, the data presented suggest that p53 may affect directly the initiation or maintenance of replicative DNA synthesis.     

Elevated c-myc expression facilitates the replication of SV40 DNA in human lymphoma cells

The v-myc oncogene can induce tumours in haematopoietic, mesenchymal and epithelial tissues. The corresponding c-myc proto-oncogene can contribute to the genesis and/or the progression of an equally wide variety of tumours when activated by retroviral insertions, chromosomal translocations or gene amplification. The c-myc gene product is a DNA-binding, nuclear phosphoprotein that is involved in the control of cell proliferation and possibly in DNA synthesis. The replication of Simian virus 40 (SV40) is a useful model system to study eukaryotic DNA replication as the virus relies almost entirely on cellular DNA replication apparatus. The SV40-based vector, pSVEpR4, replicates poorly in the human BJAB lymphoma line and in most human cells, but replicates well in Burkitt lymphoma lines, which have fused immunoglobulin and c-myc genes, resulting in high c-myc expression. Cotransfection of the BJAB cells with a c-myc-expressing construct (pI4-P6) increased the replication of pSVEpR4 tenfold. Our findings indicate that overexpression of the c-myc gene product allows the replication of SV40 in human lymphoma cells, suggesting that c-myc is involved in the control of replication.     

Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast

Cyclin-dependent kinases (CDKs) drive major cell cycle events including the initiation of chromosomal DNA replication. We identified two S phase CDK (S-CDK) phosphorylation sites in the budding yeast Sld3 protein that, together, are essential for DNA replication. Here we show that, when phosphorylated, these sites bind to the amino-terminal BRCT repeats of Dpb11. An Sld3-Dpb11 fusion construct bypasses the requirement for both Sld3 phosphorylation and the N-terminal BRCT repeats of Dpb11. Co-expression of this fusion with a phospho-mimicking mutant in a second essential CDK substrate, Sld2, promotes DNA replication in the absence of S-CDK. Therefore, Sld2 and Sld3 are the minimal set of S-CDK targets required for DNA replication. DNA replication in cells lacking G1 phase CDK (G1-CDK) required expression of the Cdc7 kinase regulatory subunit, Dbf4, as well as Sld2 and Sld3 bypass. Our results help to explain how G1- and S-CDKs promote DNA replication in yeast.     

CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast

In eukaryotic cells, cyclin-dependent kinases (CDKs) have an important involvement at various points in the cell cycle. At the onset of S phase, active CDK is essential for chromosomal DNA replication, although its precise role is unknown. In budding yeast (Saccharomyces cerevisiae), the replication protein Sld2 (ref. 2) is an essential CDK substrate, but its phospho-mimetic form (Sld2-11D) alone neither affects cell growth nor promotes DNA replication in the absence of CDK activity, suggesting that other essential CDK substrates promote DNA replication. Here we show that both an allele of CDC45 (JET1) and high-copy DPB11, in combination with Sld2-11D, separately confer CDK-independent DNA replication. Although Cdc45 is not an essential CDK substrate, CDK-dependent phosphorylation of Sld3, which associates with Cdc45 (ref. 5), is essential and generates a binding site for Dpb11. Both the JET1 mutation and high-copy DPB11 by-pass the requirement for Sld3 phosphorylation in DNA replication. Because phosphorylated Sld2 binds to the carboxy-terminal pair of BRCT domains in Dpb11 (ref. 4), we propose that Dpb11 connects phosphorylated Sld2 and Sld3 to facilitate interactions between replication proteins, such as Cdc45 and GINS. Our results demonstrate that CDKs regulate interactions between BRCT-domain-containing replication proteins and other phosphorylated proteins for the initiation of chromosomal DNA replication; similar regulation may take place in higher eukaryotes.     

Nuclear factor III, a novel sequence-specific DNA-binding protein from HeLa cells stimulating adenovirus DNA replication

Dissection and reconstitution of the adenovirus DNA replication machinery has led to the discovery of two HeLa nuclear proteins which are required in conjunction with three viral proteins. One of these, nuclear factor I (NF-I), recognizes an internal region of the origin between nucleotides 25 and 40 and by binding to one side of the helix stimulates the initiation reaction up to 30-fold. NFI-binding sites have been observed upstream of several cellular genes, such as chicken lysozyme, human IgM and human c-myc, and coincide in most cases with DNase I hypersensitive regions. Here we report the identification of a novel DNA-binding protein from HeLa nuclei, designated NF-III, that recognizes a sequence in the adenovirus origin very close to the NFI-binding site, between nucleotides 36 and 54. This sequence includes the partially conserved nucleotides TATGATAATGAG. NF-III stimulates DNA replication four- to sixfold by increasing the initiation efficiency. Potential cellular binding sites include promoter elements of the histone H2B gene, the human interferon beta gene, the human and mouse immunoglobulin VK and VH genes and the mammal/chicken/Xenopus laevis U1 and U2 small nuclear RNA genes. Furthermore, a subset of the herpes simplex virus immediate early promoter specific TAATGARAT elements is homologous with the adenovirus 2 (Ad-2) NFIII-binding site.     

Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint

The checkpoint kinase proteins Mec1 and Rad53 are required in the budding yeast, Saccharomyces cerevisiae, to maintain cell viability in the presence of drugs causing damage to DNA or arrest of DNA replication forks. It is thought that they act by inhibiting cell cycle progression, allowing time for DNA repair to take place. Mec1 and Rad53 also slow S phase progression in response to DNA alkylation, although the mechanism for this and its relative importance in protecting cells from DNA damage have not been determined. Here we show that the DNA-alkylating agent methyl methanesulphonate (MMS) profoundly reduces the rate of DNA replication fork progression; however, this moderation does not require Rad53 or Mec1. The accelerated S phase in checkpoint mutants, therefore, is primarily a consequence of inappropriate initiation events. Wild-type cells ultimately complete DNA replication in the presence of MMS. In contrast, replication forks in checkpoint mutants collapse irreversibly at high rates. Moreover, the cytotoxicity of MMS in checkpoint mutants occurs specifically when cells are allowed to enter S phase with DNA damage. Thus, preventing damage-induced DNA replication fork catastrophe seems to be a primary mechanism by which checkpoints preserve viability in the face of DNA alkylation.     

A common function for polyoma virus large-T and papillomavirus E1 proteins?

Nucleotide sequencing has revealed a common genetic organization for three papillomaviruses: BPV-1 (bovine papillomavirus type 1), HPV-1 (human papillomavirus type 1a) and HPV-6 (human papillomavirus type 6b). Several open reading frames, corresponding to as yet uncharacterized proteins, were observed in these genomes in the region that is required for oncogenic transformation by BPV-1 and for plasmidial maintenance of its genome. The longest of these frames, E1, is also the most conserved between the three viruses; we have compared the amino acid sequence of its putative product ('E1 protein') with those of the large-T proteins of three polyoma viruses and report here significant homologies in their carboxy-terminal halves, extending for over 200 amino acids. Moreover, similar secondary structures were predicted in this region, especially in two blocks of homologous residues, which correspond in the large-T proteins of polyoma and simian virus 40 (SV40) viruses to sites involved in the ATPase and nucleotide-binding activities. These observations suggest that the papillomavirus E1 proteins might have a function in common with the polyoma virus large-T proteins (which are required for the initiation of viral DNA replication). As it was suggested recently that the E1 gene product is involved in maintaining the BPV-1 genome as a plasmid in transformed cells, we speculate that the structural features conserved in these otherwise very different viruses are general characteristics of eukaryotic proteins involved in the control of DNA replication.