Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis.
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Abstract:
We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.Keywords:
Chinese hamster
Replication factor C
Temperature-sensitive mutant
DNA polymerase II
Replication factor C
Origin recognition complex
Pre-replication complex
Licensing factor
Ter protein
DNA polymerase delta
Replication protein A
Minichromosome maintenance
S phase
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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.
Replication factor C
DNA polymerase delta
Origin recognition complex
DNA polymerase II
Replication protein A
DNA clamp
Primer (cosmetics)
Pre-replication complex
Ter protein
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Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.
Replication factor C
Origin recognition complex
DNA polymerase II
DNA polymerase delta
Primase
DNA clamp
Replication protein A
Pre-replication complex
Ter protein
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Origin recognition complex
Replication factor C
Pre-replication complex
Licensing factor
Minichromosome maintenance
S phase
DNA re-replication
Replication protein A
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Replication factor A (RF-A) is a multisubunit, cellular protein that functions with SV40 T antigen during the initiation stage of DNA replication at the SV40 origin. It also cooperates with other replication factors to stimulate the activity of both polymerases alpha and delta during chain elongation. RF-A from both human and yeast cells is phosphorylated in a cell-cycle-dependent manner; the protein is phosphorylated at the G1- to S-phase transition, and dephosphorylation occurs at mitosis, thereby resetting this cycle. This observation provides a direct link between a protein required for DNA replication and cell-cycle-regulated protein phosphorylation.
Replication factor C
Origin recognition complex
S phase
Pre-replication complex
DNA re-replication
Licensing factor
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ABSTRACT A major function of the DNA damage responses (DDRs) that act during the replicative phase of the cell cycle is to inhibit initiation and elongation of DNA replication. The polyomavirus SV40 is an important model system for studying human DNA replication and DDRs due to its heavy reliance on host factors for viral DNA replication, and the arrest of SV40 DNA replication in response to DDR activation. The inhibition of SV40 DNA replication following DDR activation is associated with enhanced DDR kinase phosphorylation of SV40 Large T-antigen (LT), the viral origin-binding protein and DNA helicase. NetPhos prediction of LT phosphorylation on multiple sites were confirmed by mass spectroscopy, including a highly conserved DDR kinase site, T518. In cell-based DNA replication assays expression of the phosphomimetic mutant form of LT at T518 (T518D) resulted in dramatically decreased levels of SV40 DNA replication; while LT-dependent transcriptional activation was unaffected. WT and LT T518D were subsequently expressed, purified, and analyzed in vitro for assessment of biochemical function. In concordance with the cell-based data, reactions using SV40 LT-T518D, but not T518A, showed dramatic inhibition of SV40 DNA replication. Importantly, the LT T518D mutation did not affect critical LT protein interactions or its ATPase function, but showed decreased helicase activity on long, but not very short, DNA templates. These results suggest that DDR phosphorylation at T518 inhibits SV40 DNA replication by impeding LT helicase activity, thereby slowing the DNA replication fork. This is consistent with the slowing of cellular replication forks following DDR and may provide a paradigm for another mechanism for how DNA replication forks can be slowed in response to DDR, by phosphorylation of DNA helicases.
Origin recognition complex
Replication factor C
S phase
Pre-replication complex
Replication protein A
Minichromosome maintenance
dnaB helicase
DNA polymerase delta
DnaA
DNA polymerase II
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In vitro replication of papillomavirus DNA has been carried out with a combination of purified proteins and partially purified extracts made from human cells. DNA synthesis requires the viral E1 protein and the papillomavirus origin of replication. The E2 protein stimulates DNA synthesis in a binding site-independent manner. Papillomavirus DNA replication is also dependent on the cellular factors replication protein A, replication factor C, and proliferating-cell nuclear antigen as well as a phosphocellulose column fraction (IIA). Fraction IIA contains DNA polymerase alpha-primase and DNA polymerase delta. Both of these polymerases are essential for papillomavirus DNA replication in vitro. However, unlike the case with T-antigen-dependent replication from the simian virus 40 origin, purified DNA polymerase alpha-primase and delta cannot efficiently replace fraction IIA in the replication reaction. Hence, additional cellular factors seem to be required for papillomavirus DNA replication. Interestingly, replication factor C and proliferating-cell nuclear antigen are more stringently required for DNA synthesis in the papillomavirus system than in the simian virus 40 in vitro system. These distinctions indicate that there must be mechanistic differences between the DNA replication systems of papillomavirus and simian virus 40.
Replication factor C
DNA polymerase delta
DNA polymerase II
Origin recognition complex
Primase
DNA clamp
Bovine papillomavirus
Pre-replication complex
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In the presence of large T antigen and plasmids containing a functional origin of replication, extracts from a human cell line will support multiple rounds of simian virus 40 (SV40) replication in vitro. Fractionation of this extract has led to the identification of several factors, some of which have been purified to homogeneity. The characterisation of these proteins has led to the separation of SV40 replication in vitro into multiple stages. Two proteins, the cell cycle-regulated proliferating cell nuclear antigen and replication factor-C, have been shown to be essential for coordinating leading and lagging strand synthesis in this system. Another protein, replication factor-A, is a multi-subunit protein of 70, 34 and 11K (K = 10(3) Mr) polypeptides which, because of its high affinity for DNA, is thought to function as a eukaryotic single-stranded DNA binding protein. Interactions between other cellular factors are also described that effect the initiation of DNA replication, but are not required in a more purified system. In addition a model for a hypothetical replication fork is described, which suggests a role for both alpha- and delta-polymerases in this system, and may be applicable to higher eukaryotes.
Replication factor C
Origin recognition complex
Pre-replication complex
Licensing factor
Ter protein
S phase
Minichromosome maintenance
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The replication of DNA in the eukaryotic cell cycle is one of the most highly regulated events in cell growth and division. Biochemical studies on the replication of the genome of the small DNA virus simian virus 40 (SV40) have resulted in the identification of a number of DNA replication proteins from human cells. One of these, Replication Protein A (RPA), was phosphorylated in a cell cycle-dependent manner, beginning at the onset of DNA replication. RPA was phosphorylated in vitro by the cell cycle-regulated cdc2 protein kinase. This kinase also stimulated the unwinding of the SV40 origin of DNA replication during initiation of DNA replication in vitro, suggesting a mechanism by which cdc2 kinase may regulate DNA replication. Functional homologues of the DNA replication factors have been identified in extracts from the yeast Saccharomyces cerevisiae, enabling a genetic characterization of the role of these proteins in the replication of cellular DNA. A cellular origin binding protein had not been characterized. To identify proteins that function like T antigen at cellular origins of DNA replication, we examined the structure of a yeast origin of DNA replication in detail. This origin consists of four separate functional elements, one of which is essential. A multiprotein complex that binds to the essential element has been identified and purified. This protein complex binds to all known cellular origins from S. cerevisiae and may function as an origin recognition complex.
Origin recognition complex
Pre-replication complex
Replication factor C
Licensing factor
S phase
Replication protein A
Minichromosome maintenance
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During the mitotic mammalian cell cycle cells faithfully replicate their DNA utilizing multiple DNA replication sites known as origins of replication. DNA is replicated to provide each daughter cell a complete copy of the genome. Replication proceeds bi-directionally from a minority of potential origins licensed for replication by a variety of replication factor proteins. Replication is catalysed by processive replication enzymes known as DNA polymerases and is limited to the synthesis phase (S phase) of the cell cycle. Changes in the timing of replication, origin usage and replication rate are indicative of DNA replication stress, a proposed hallmark of cancer that causes genome instability. Cell cycle progression is largely controlled by the activity of cyclin dependent kinases (CDKs) and their cyclin binding partners. Here using an in vitro cell-free DNA replication system we analyse the interplay between Ciz1 and cyclin A/CDK2 in regulation of the initiation phase of DNA replication. This demonstrates that Ciz1 modulates and enhances the activity of cyclin A-CDK2 in cell free DNA replication assays and that Ciz1 increases the permissive CDK range that can promote DNA replication. Next the inhibitory effect of Ap4A in cell free DNA replication assays is studied. These data suggest that Ap4A inhibits initiation by reducing loading of the replicative helicase MCM2-7 and the DNA polymerase sliding clamp PCNA. These data suggest that Ap4A can inhibit the firing of replication origins through disruption of replication complex assembly. Finally, DNA combing is established to measure replication parameters. Here we find that the replication fork progresses at 1.3kbp/min in mouse fibroblast cells, consistent with other studies, and quantify replication fork stalling by replication inhibitor aphidicolin. These data demonstrate the potential for cell free DNA replication assays to be combined with DNA combing to dissect replication parameters and characterise DNA replication stress in future studies.
Origin recognition complex
Replication factor C
Pre-replication complex
S phase
Licensing factor
Minichromosome maintenance
DNA re-replication
DNA polymerase delta
Replication protein A
Semiconservative replication
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