DNA repair deficiency and BPDE-induced chromosomal alterations in CHO cells

The induction of chromosomal aberrations and sister chromatid exchanges by BPDE was evaluated in parental and different DNA repair deficient Chinese hamster ovary cell lines in order to elucidate the mechanisms involved in their induction. These included the parental line (AA8), nucleotide excision repair (UV4, UV5, UV61), base excision repair (EM9), homologous recombination repair (Irs1SF) and non-homologous end joining (V3-3) deficient ones. The ranking of different cell lines for BPDE-induced chromosome aberrations was: UV4, Irs1SF, UV5, UV 61, EM9, V3-3, and AA8 in a descending order. Cells deficient in NER and HRR were found to be very sensitive, indicating the importance of these pathways in the repair of lesions induced by BPDE. For induction of SCEs, HRR and BER deficient cells were refractory, whereas the other cell lines responded with a dose-dependent increase. The possible mechanisms involved in BPDE-induced chromosomal alterations are discussed.     

The initiation of chromosomal DNA replication in eukaryotes

Eukaryotic DNA replication initiates at many sites on each chromosome during the S phase of the cell cycle. Each origin of replication lies in a unique chromosomal environment and can be regulated in different cell types both at the level of utilization and the time of initiation during S phase. In this review, we examine the control and the mechanism of eukaryotic origin function.     

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.     

Bromodeoxyuridine/DNA analysis of replication in CHO cells after exposure to UV light

The effects of ultraviolet light on cellular DNA replication were evaluated in an asynchronous Chinese hamster ovary cell population. BrdUrd incorporation was measured asa function of cell-cycle position, using an antibody against bromodeoxyuridine (BrdUrd) and dual parameter flow cytometric analysis. After exposure to UV light, there was an immediate reduction ( 50%) of BrdUrd incorporation in S phase cells, with most of the cells of the population being affected to a similar degree. At 5 h after UV, a population of cells with increased BrdUrd appeared as cells that were in G1 phase at the time of irradiation entered S phase with apparently increased rates of DNA synthesis. For 8 h after UV exposure, incorporation of BrdUrd by the original S phase cells remained constant, whereas a significant portion of original G1 cells possessed rates of BrdUrd incorporation surpassing even those of control cells. Maturation rates of DNA synthesized immediately before or after exposure by alkaline elution, were similar. Therefore, DNA synthesis measured in the short pulse by anti-BrdUrd fluorescence after exposure to UV light was representative of genomic replication. Anti-BrdUrd measurements after DNA damage provide quantitative and qualitative information of cellular rates of DNA synthesis especially in instances where perturbation of cell-cycle progression is a dominant feature of the damage. In this study, striking differences of subsequent DNA synthesis rates between cells in G1 or S phase at the time of exposure were revealed.     

Mre11 protein complex prevents double-strand break accumulation during chromosomal DNA replication

Mre11 complex promotes repair of DNA double-strand breaks (DSBs). Xenopus Mre11 (X-Mre11) has been cloned, and its role in DNA replication and DNA damage checkpoint studied in cell-free extracts. DSBs stimulate the phosphorylation and 3'-5' exonuclease activity of X-Mre11 complex. This induced phosphorylation is ATM independent. Phosphorylated X-Mre11 is found associated with replicating nuclei. X-Mre11 complex is required to yield normal DNA replication products. Genomic DNA replicated in extracts immunodepleted of X-Mre11 complex accumulates DSBs as demonstrated by TUNEL assay and reactivity to phosphorylated histone H2AX antibodies. In contrast, the ATM-dependent DNA damage checkpoint that blocks DNA replication initiation is X-Mre11 independent. These results strongly suggest that the function of X-Mre11 complex is to repair DSBs that arise during normal DNA replication, thus unraveling a critical link between recombination-dependent repair and DNA replication.     

Contribution of DNA polymerase delta to DNA replication in permeable CHO cells synchronized in S phase.

To evaluate the relative contributions of DNA polymerase alpha and DNA polymerase delta in chromosome replication during the S phase of the cell cycle, we have used the permeable cell system for replication as a functional assay. We carried out the analysis of DNA polymerases both in quiescent cells stimulated to proliferate and progress through the cell cycle (monolayers) and in actively growing cells separated into progressive stages of the cell cycle by centrifugal elutriation (suspension cultures). DNA polymerase alpha was measured by using the inhibitor butylphenyl dGTP at low concentrations. Using several inhibitors such as aphidicolin, ddTTP and butylphenyl dGTP, we found that DNA polymerase alpha and delta activity were coordinately increased during S phase and declined at the end. However, DNA polymerase delta was performing about 80% of the total replication and DNA polymerase alpha performed only 20%. This high ratio of DNA polymerase delta to DNA polymerase alpha replication activity was maintained throughout S phase in two entirely different experimental approaches.     

The rate of fork movement during DNA replication in mammalian cells

DNA fiber autoradiography was used to measure the rate of replication fork progression along replication units in human diploid cells. The rate in different replication units differs very significantly and lies within the range 0.1 to 1.2 m/min. However, no significant changes were found in the rate of fork movement along single replication units operating during long intervals of S phase. Moreover, the fork progression rate is constant in many replication units of human cells.     

GINS is a DNA polymerase epsilon accessory factor during chromosomal DNA replication in budding yeast

GINS is a protein complex found in eukaryotic cells that is composed of Sld5p, Psf1p, Psf2p, and Psf3p. GINS polypeptides are highly conserved in eukaryotes, and the GINS complex is required for chromosomal DNA replication in yeasts and Xenopus egg. This study reports purification and biochemical characterization of GINS from Saccharomyces cerevisiae. The results presented here demonstrate that GINS forms a 1:1 complex with DNA polymerase epsilon (Pol epsilon) holoenzyme and greatly stimulates its catalytic activity in vitro. In the presence of GINS, Pol epsilon is more processive and dissociates more readily from replicated DNA, while under identical conditions, proliferating cell nuclear antigen slightly stimulates Pol epsilon in vitro. These results strongly suggest that GINS is a Pol epsilon accessory protein during chromosomal DNA replication in budding yeast. Based on these results, we propose a model for molecular dynamics at eukaryotic chromosomal replication fork.     

Intermediates of chromosomal DNA replication in Escherichia coli

The product of bacteriophage T4 gene 63 has two activities, one which catalyzes the attachment of tail fibers to base plates during morphogenesis (TFA) and one which catalyzes the joining of single-stranded polynucleotides (RNA ligase). The only phenotype attributed to mutations in gene 63 is a defect in attachment of tail fibers leading to fiberless T4 particles. However, it is suspected that TFA and RNA ligase are unrelated activities of the same protein since they have very different requirements in vitro.We have isolated new mutants which have lost the RNA ligase but have retained the TFA activity of the product of gene 63. These mutants exhibit defects in T4 DNA replication and late gene expression in some strains of Escherichia coli. This work allows us to draw three conclusions: (1) the TFA and RNA ligase activities are unrelated functions of the gene 63 product making this the prototype for a protein which has more than one unrelated function; (2) the RNA ligase is probably involved in DNA metabolism rather than RNA processing as has been proposed: (3) the RNA ligase and polynucleotide 5′ kinase 3′ phosphatase of T4 perform intimately related functions.     

Plasmid and chromosomal DNA replication and partitioning during the Caulobacter crescentus cell cycle

Cell division in Caulobacter crescentus yields a swarmer and a stalked cell. Only the stalked cell progeny is able to replicate its chromosome, and the swarmer cell progeny must differentiate into a stalked cell before it too can replicate its chromosome. In an effort to understand the mechanisms that limit chromosomal replication to the stalked cell, plasmid DNA synthesis was analyzed during the developmental cell cycle of C. crescentus, and the partitioning of both the plasmids and the chromosomes to the progeny cells was examined. Unlike the chromosome, plasmids from the incompatibility groups Q and P replicated in all C. crescentus cell types. However, all plasmids tested showed a ten- to 20-fold higher replication rate in the stalked cells than the swarmer cells. We observed that all plasmids replicated during the C. crescentus cell cycle with comparable kinetics of DNA synthesis, even though we tested plasmids that encode very different known (and putative) replication proteins. We determined the plasmid copy number in both progeny cell types, and determined that plasmids partitioned equally to the stalked and swarmer cells. We also reexamined chromosome partitioning in a recombination-deficient strain of C. crescentus, and confirmed an earlier report that chromosomes partition to the progeny stalked and swarmer cells in a random manner that does not discriminate between old and new DNA strands.     

The relationship between sister-chromatid exchange and perturbations in DNA replication in mutant EM9 and normal CHO cells

The majority of the high (12-fold elevated) baseline sister-chromatid exchanges (SCEs) that occur in the CHO mutant line EM9 appear to be a consequence of incorporated BrdUrd, and they arise during replication of DNA containing BrdUrd in a template strand. In normal CHO cells the alkaline elution patterns of DNA newly replicated on a BrdUrd-containing template are significantly altered compared with those seen during the replication on an unsubstituted template. The nascent DNA synthesized on such an altered template is delayed in reaching mature size, possibly because replication forks are temporarily blocked at sites occurring randomly along the template. Transient blockage of replication forks may be a prerequisite for SCE. The delay in replication on BrdUrd-substituted templates was greater in EM9 cells than in parental AA8 cells and was also greater in AA8 cells treated with benzamide, an inhibitor of poly(ADPR) polymerase, than in untreated AA8 cells. Under these conditions, treatment with benzamide also produced a 7-fold increase in SCEs in AA8. An EM9-derived revertant line that has a low baseline SCE frequency showed less delay in replication on BrdUrd-substituted templates than did EM9. However, under conditions where the template strand contained CldUrd, which was shown to produce 4-fold more SCEs than BrdUrd in AA8 cells, the replication delay in AA8 was not any greater in the CldUrd-substituted cells. Thus, other factors besides the delay appear to be involved in the production of SCEs by the template lesions resulting from incorporation of the halogen-substituted pyrimidine molecules.     

Chromatin assembly during DNA replication in somatic cells.

Newly replicated DNA is assembled into chromatin through two principle pathways. Firstly, parental nucleosomes segregate to replicated DNA, and are transferred directly to one of the two daughter strands during replication fork passage. Secondly, chromatin assembly factors mediate de-novo assembly of nucleosomes on replicating DNA using newly synthesized and acetylated histone proteins. In somatic cells, chromatin assembly factor 1 (CAF-1) appears to be a key player in assembling new nucleosomes during DNA replication. It provides a molecular connection between newly synthesized histones and components of the DNA replication machinery during the S phase of the cell division cycle.