Normal uracil-DNA glycosylase activity in Bloom's syndrome cells

Cells from patients with Bloom's syndrome, a rare human disease with autosomal recessive mode of inheritance, exhibit cytological abnormalities involving DNA metabolism. Bloom's syndrome is characterized by a greatly increased cancer frequency which may reflect a specific defect in DNA repair and replication. Evidence has recently been presented of the existence in Bloom's syndrome of an abnormality of the DNA ligase involved in semiconservative DNA replication. Another abnormality, in the excision-repair pathway of Bloom's syndrome cells, is reportedly due to an aberrant immunological reactivity of the DNA-repair enzyme uracil-DNA glycosylase. In this investigation we show, however, that the catalytic activity of uracil-DNA glycosylase appears to be normal in Bloom's syndrome lymphoblastoid cells.     

FANCJ couples replication past natural fork barriers with maintenance of chromatin structure

Defective DNA repair causes Fanconi anemia (FA), a rare childhood cancer–predisposing syndrome. At least 15 genes are known to be mutated in FA; however, their role in DNA repair remains unclear. Here, we show that the FANCJ helicase promotes DNA replication in trans by counteracting fork stalling on replication barriers, such as G4 quadruplex structures. Accordingly, stabilization of G4 quadruplexes in ΔFANCJ cells restricts fork movements, uncouples leading- and lagging-strand synthesis and generates small single-stranded DNA gaps behind the fork. Unexpectedly, we also discovered that FANCJ suppresses heterochromatin spreading by coupling fork movement through replication barriers with maintenance of chromatin structure. We propose that FANCJ plays an essential role in counteracting chromatin compaction associated with unscheduled replication fork stalling and restart, and suppresses tumorigenesis, at least partially, in this replication-specific manner.     

DNA replication during a serum-induced S phase in primate CV-1 cells

We have investigated components of DNA replication in a serum-induced S phase of primate CV-1 cells. Using DNA fiber autoradiography, we found a relative decrease in the frequency of initiation events in mid-S compared with early and late S phase. The other components of DNA replication measured by autoradiography—synchrony of initiation events, size of replication units, incidence of bidirectional replication, and the rate of replication fork movement—remained constant through S phase. When fork movement was measured by density gradient analysis of BUdR- and [³H]-thymidine-substituted DNA, it was also found to remain constant. These results show that most components of DNA replication are invariable through a serum-induced S phase. The changes in initiation frequency support the view that it may be critical in the regulation of ongoing replication.     

The interrelation between changes in the structural organization of replicon clusters, a retarded fork displacement rate and the high level of spontaneous SCEs in form II of xeroderma pigmentosum]

A cytogenetic observation, that the sister chromatid exchanges (SCE) occur 3 times more frequently in a special form of xeroderma pigmentosum--XPII than in the norm, prompted a study of DNA replication in this rare disease. Using DNA fiber autoradiography, the rate of fork movement and the frequency of initiation in the adjacent clusters of replicons were estimated. The rate of fork movement was significantly slower than that in classical XP and in normal cells. Here evidence was provided on another defect in DNA replication in XPII that involves a significantly decreased number of simultaneously operating adjacent clusters of replicons, which results in a decreased rate of DNA chain-growth. According to the Painter replication model for SCE, the exchanges arise due to double-strand DNA breaks occurring on the border between two adjacent clusters, respectively, completely and partially replicated. A retarded fork-displacement rate together with a decreased rate of DNA-chain growth may cause this situation to persist longer than in the norm. Thus, our data provide a further support of the replication model for SCE. A similar combination of cytogenetic and molecular defects has been obtained earlier in the Bloom syndrome cells.     

The action of ionizing radiation on DNA replication in the cells of a healthy human subject and of a patient with Down's syndrome

Using DNA fiber autoradiography we have revealed a new defect earlier unknown in Down's syndrome but analogous to that earlier shown by the authors in AT and basal cell nevus. This syndrome involves a significantly decreased number of simultaneously operating groups of replicons compared to that in normal cells., the rate of fork movement and the fusion of neighbouring units in the group being unchanged. Ionizing radiation (5 Gy) does not change the rate of DNA chain growth in the cells derived from the affected individuals, however, it significantly reduces this parameter in normal cells due to inhibition of replicon initiation in the whole clusters. Thus, radioresistant DNA synthesis in chromosomal instability syndromes may be explained by some defect in DNA replication in unirradiated cells analogous to that in irradiated normal cells.     

Visualization of altered replication dynamics after DNA damage in human cells

Eukaryotic cells respond to DNA damage within the S phase by activating an intra-S checkpoint: a response that includes reducing the rate of DNA synthesis. In yeast cells this can occur via checkpoint-dependent inhibition of origin firing and stabilization of ongoing forks, together with a checkpoint-independent slowing of fork movement. In higher eukaryotes, however, the mechanism by which DNA synthesis is reduced is less clear. We have developed strategies based on DNA fiber labeling that allow the quantitative assessment of rates of replication fork movement, origin firing, and fork stalling throughout the genome by examining large numbers of individually labeled replication forks. We show that exposing S phase cells to ionizing radiation induces a transient block to origin firing but does not affect fork rate or fork stalling. Alkylation damage by methyl methane sulfonate causes a slowing of fork movement and a high rate of fork stalling, in addition to inducing a block to new origin firing. Nucleotide depletion by hydroxyurea also reduces replication fork rate and increases stalling; moreover, in contrast to a recent report, we show that hydroxyurea induces a strong block to new origin firing. The DNA fiber labeling strategy provides a powerful new approach to analyze the dynamics of DNA replication in a perturbed S phase.     

Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system

Initiation of DNA replication is tightly controlled during the cell cycle to maintain genome integrity. In order to directly study this control we have previously established a cell-free system from human cells that initiates semi-conservative DNA replication. Template nuclei are isolated from cells synchronized in late G₁ phase by mimosine. We have now used DNA combing to investigate initiation and further progression of DNA replication forks in this human in vitro system at single molecule level. We obtained direct evidence for bidirectional initiation of divergently moving replication forks in vitro. We assessed quantitatively replication fork initiation patterns, fork movement rates and overall fork density. Individual replication forks progress at highly heterogeneous rates (304 ± 162 bp/min) and the two forks emanating from a single origin progress independently from each other. Fork progression rates also change at the single fork level, suggesting that replication fork stalling occurs. DNA combing provides a powerful approach to analyse dynamics of human DNA replication in vitro.     

RecD2 Helicase Limits Replication Fork Stress in Bacillus subtilis

DNA helicases have important roles in genome maintenance. The RecD helicase has been well studied as a component of the heterotrimeric RecBCD helicase-nuclease enzyme important for double-strand break repair in Escherichia coli. Interestingly, many bacteria lack RecBC and instead contain a RecD2 helicase, which is not known to function as part of a larger complex. Depending on the organism studied, RecD2 has been shown to provide resistance to a broad range of DNA-damaging agents while also contributing to mismatch repair (MMR). Here we investigated the importance of Bacillus subtilis RecD2 helicase to genome integrity. We show that deletion of recD2 confers a modest increase in the spontaneous mutation rate and that the mutational signature in ΔrecD2 cells is not consistent with an MMR defect, indicating a new function for RecD2 in B. subtilis. To further characterize the role of RecD2, we tested the deletion strain for sensitivity to DNA-damaging agents. We found that loss of RecD2 in B. subtilis sensitized cells to several DNA-damaging agents that can block or impair replication fork movement. Measurement of replication fork progression in vivo showed that forks collapse more frequently in ΔrecD2 cells, supporting the hypothesis that RecD2 is important for normal replication fork progression. Biochemical characterization of B. subtilis RecD2 showed that it is a 5′-3′ helicase and that it directly binds single-stranded DNA binding protein. Together, our results highlight novel roles for RecD2 in DNA replication which help to maintain replication fork integrity during normal growth and when forks encounter DNA damage.     

Replication of polyoma DNA in isolated nuclei: analysis of replication fork movement.

The movement of replication forks during polyoma DNA synthesis in isolated nuclei was analyzed by digesting newly synthesized DNA with the restriction endonuclease HpaII which cleaves polyoma DNA into eight unique fragments. The terminus of in vitro DNA synthesis was identified by cleaving newly completed molecules with HpaII. The distribution of label in the restriction fragments showed that the in vitro DNA synthesis was bidirectional and had the normal terminus of replication. Analysis of replicative intermediates pulse-labeled in vitro further suggested that DNA synthesis in isolated nuclei is an ordered process similar to replication in intact cells. Replication forks moved with a constant rate from the origin towards the terminus of replication. The nonlinear course of the DNA synthesis reaction in the isolated nuclei seems to result from the random inactivation of replication forks rather than a decrease in the rate of fork movement. During the in vitro synthesis a replication fork could maximally synthesize a DNA chain about 1,000 nucleotides long. The results suggest that some replication forks might be initiated in vitro at the origin of replication.     

Chk1 requirement for high global rates of replication fork progression during normal vertebrate S phase

Chk1 protein kinase maintains replication fork stability in metazoan cells in response to DNA damage and DNA replication inhibitors. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which Chk1 maintains global replication fork rates during normal vertebrate S phase. We report that replication fork rates in Chk1^−/− chicken DT40 cells are on average half of those observed with wild-type cells. Similar results were observed if Chk1 was inhibited or depleted in wild-type DT40 cells or HeLa cells by incubation with Chk1 inhibitor or small interfering RNA. In addition, reduced rates of fork extension were observed with permeabilized Chk1^−/− cells in vitro. The requirement for Chk1 for high fork rates during normal S phase was not to suppress promiscuous homologous recombination at replication forks, because inhibition of Chk1 similarly slowed fork progression in XRCC3^−/− DT40 cells. Rather, we observed an increased number of replication fibers in Chk1^−/− cells in which the nascent strand is single-stranded, supporting the idea that slow global fork rates in unperturbed Chk1^−/− cells are associated with the accumulation of aberrant replication fork structures.     

O6-methylguanine methyltransferase increases before S phase in normal human cells but does not increase in hypermutable Bloom's syndrome cells

The regulation of the O6-methylguanine methyltransferase was examined during cell proliferation in hypermutable Bloom's syndrome fibroblasts and normal human skin fibroblasts. During synchronous growth following serum stimulation normal human cells enhanced methyltransferase activity 2.4-fold in the absence of exogenous damage as a normal regulatory event during the cell cycle. Methyltransferase activity was increased prior to the induction of DNA replication and of DNA polymerase and was diminished when each replicative activity was maximal. In contrast, although methyltransferase levels in quiescent cells are equivalent, hypermutable Bloom's syndrome cells did not increase methyltransferase at any interval in the cell cycle.     

Disruption of DNA replication in non-irradiated cells in basal cell nevus syndrome and the effect of ionizing radiation

Analysis of DNA fiber autoradiograms from basal cell nevus syndrome (BCNS) skin fibroblasts has revealed for the first time a new defect in DNA replication earlier unknown in other chromosomal instability syndromes, that involves a significantly decreased rate of DNA-chain growth in unirradiated cells. Here we present evidence that the defect may be due to a marked reduction in number of simultaneously operating groups of replicons compared to that in normal cells, the rate of fork movement and the fusion of neighbouring units in the group remaining unchanged. Radioresistant DNA synthesis was observed in the BCNS cells. The exposure of cells derived from normal donor to gamma-rays at a dose of 5 Gy reduces the number of simultaneously operating groups of replicons to the level occurring in unirradiated BCNS cells, the rate of folk movement being unchanged in both cell types. However, the incidence of fusion between neighbouring units within the group is lower in the cells exposed to gamma-rays, due perhaps to a radiation-induced lesion in the group. Thus, ionizing radiation reduces the rate of DNA synthesis to the same level, however from different initial levels. Our data suggest that the phenomenon of radioresistant DNA synthesis may be explained by the presence of the initial defect in DNA replication in BCNS or any other chromosomal instability disorders.     

Stimulation of deoxyribonucleic acid replication fork movement by spermidine analogs in polyamine-deficient Escherichia coli.

We examined the rate of deoxyribonucleic acid (DNA) replication fork movement in polyamine-deficient cells of Escherichia coli by two independent techniques. DNA autoradiography was used to directly visualize the length of DNA produced during a given time interval, and replication rates were calculated. The amount of DNA synthesized after blocking protein synthesis also allowed calculation of replication rates. We found that the DNA chain elongation rate in polyamine-deficient cells was about half that of putrescine- or spermidine-supplemented cells. We also found that spermidine homologs of increasing chain length, when present at equal intracellular concentrations, exhibited a decreasing ability to support growth and the rate of DNA replication fork movement. The kinetics of recovery of DNA synthesis from the polyamine-deficient state were also investigated. A new rate of DNA synthesis was reached about 20 min after addition of spermidine to polyamine-limited cells. The rise in the rate of DNA synthesis was preceded by a rise in the intracellular concentration of spermidine.     

The Bloom's syndrome helicase can promote the regression of a model replication fork

Homozygous inactivation of BLM gives rise to Bloom's syndrome, a disorder associated with genomic instability and cancer predisposition. BLM encodes a member of the RecQ DNA helicase family that is required for the maintenance of genome stability and the suppression of sister-chromatid exchanges. BLM has been proposed to function in the rescue of replication forks that have collapsed or stalled as a result of encountering lesions that block fork progression. One proposed mechanism of fork rescue involves regression in which the nascent leading and lagging strands anneal to create a so-called "chicken foot" structure. Here we have developed an in vitro system for analysis of fork regression and show that BLM, but not Escherichia coli RecQ, can promote the regression of a model replication fork. BLM-mediated fork regression is ATP-dependent and occurs processively, generating regressed arms of >250 bp in length. These data establish the existence of a eukaryotic protein that could promote replication fork regression in vivo and suggest a novel pathway through which BLM might suppress genetic exchanges.     

Changes in the rate of DNA replication fork movement during S phase in mammalian cells.

DNA fiber autoradiography was used to measure the rate of replication fork movement and the size of replication units as a function of time during the S phase of synchronized Chinese hamster ovary cells. The rate of fork movement increased by about threefold from early S to later S phase, with the most dramatic change occurring in the first hour of S phase. On the other hand, the size of replication units did not vary significantly during S phase.     

The Mammalian DNA Replication Elongation Checkpoint: Implication of Chk1 and Relationship with Origin Firing as Determined by Single DNA Molecule and Single Cell Analyses

The regulation of DNA replication initiation is well documented, for both unperturbed and damaged cells. The regulation of elongation, or fork velocity, however, has only recently been revealed with the advent of new techniques allowing us to view DNA replication at the single cell and single DNA molecule levels. Normally in S phase, the progression of replication forks and their stability are regulated by the ATR-Claspin-Chk1 pathway. We recently showed that replication fork velocity varies across the human genome in normal and cancer cells, but that the velocity of a given fork is positively correlated with the distance between origins on the same DNA fiber. Accordingly, in DNA replication-deficient Bloom’s syndrome cells, reduced fork velocity is associated with an increased density of replication origins. Replication elongation is also regulated in response to DNA damage. In human colon carcinoma cells treated with the topoisomerase I inhibitor camptothecin, DNA replication is inhibited both at the level of initiation and at the level of elongation through a Chk1-dependent checkpoint mechanism. Together, these new findings demonstrate that replication fork velocity (fork progression) is coordinated with inter-origin distance and that it can be actively slowed down by Chk1-dependent mechanisms in response to DNA damage. Thus, we propose that the intra-S phase checkpoint consist of at least three elements: (1) stabilization of damaged replication forks; (2) suppression of firing of late origins; and (3) arrests of normal ongoing forks to prevent further DNA lesions by replication of a damaged DNA template.     

DNA fork displacement rates in Bloom's syndrome fibroblasts

DNA fork displacement rates were measured in three lines of Bloom's syndrome cells and in a normal diploid fibroblast line. Fork displacement rates in Bloom's cells were approx. 55–65% of the rate in normal fibroblasts.     

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.     

Functions of RecQ family helicases: possible involvement of Bloom's and Werner's syndrome gene products in guarding genome integrity during DNA replication

Escherichia coli RecQ helicase is a component of the RecF pathway of recombination whose components are required to reassemble a replisome complex at the site of the replication fork after the removal of a lesion. There are at least five RecQ homologues in human cells, including BLM and WRN. The genes encoding BLM and WRN are mutated in the cancer-prone disorder Bloom's syndrome (BS) and the plogeroid disorder Werner's syndrome (WS), respectively. These syndromes are characterized by a high degree of genomic instability, including chromosomal breaks, multiple large deletions, and translocations, and cells derived from BS and WS patients show defects in DNA replication. Recently, it has become clear that a Holliday junction-like structure is formed at stalled replication forks to result in the formation of double-stranded breaks, and recombination plays an important role in the repair of stalled or broken replication forks, leading to the reinitiation of replication. Defects in the processing of stalled replication forks could lead to aberrant recombination events resulting in genetic instability. Recent studies on BLM, WRN, and the RecQ homologue of Saccharomyces cerevisiae, Sgs1, indicate that these RecQ homologues interact with proteins involved in DNA replication, and function in a pathway from the DNA replication check point to homologous recombination.     

Immunological alteration of the Bloom's syndrome uracil DNA glycosylase in Epstein-Barr virus-transformed human lymphoblastoid cells

The immunological reactivity of the uracil DNA glycosylase was investigated in three Epstein-Barr virus-transformed human lymphoblastoid cell lines. Two were derived from normal human lymphocytes while the third was derived from a Bloom's syndrome patient. A panel of 3 anti-human placental uracil DNA glycosylase monoclonal antibodies (37.04.12, 40.10.09 and 42.08.07) was used. Immunological reactivity was determined in a double-blind enzyme-linked immunosorbent assay (ELISA); by inhibition of enzyme activity; and by immunoblot analysis. In the ELISA, the glycosylase from each lymphoblastoid cell line was recognized by glycosylase antibodies 37.04.12 and 42.08.07. In contrast, antibody 40.10.09 failed to recognize the glycosylase from the Bloom's syndrome cell line. Further analysis demonstrated that the 40.10.09 antibody was unable to inhibit catalysis by the Bloom's syndrome lymphoblast glycosylase. In contrast, the 40.10.09 antibody inhibited the activity of the two normal human lymphoblast enzymes. Denaturation of the Bloom's syndrome lymphoblast glycosylase rendered that protein immunoreactive with the 40.10.09 antibody. These results demonstrated that: (1) the immunological alteration in the Bloom's syndrome uracil DNA glycosylase was detected in hematopoietic cells; and (2) viral transformation did not affect the immunoreactivity of the enzyme from either normal human or Bloom's syndrome cells.