DNA damage in synchronized HeLa cells irradiated with ultraviolet.

The lethal effect of UV radiation of HeLa cells is least in mitosis and greatest in late G1-early S. Photochemical damage to HeLa DNA, as measured by thymine-containing dimer formation and by alkaline sucrose sedimentation, also increases from mitosis towards early S phase. Computer simulations of UV absorption by an idealized HeLa cell at different stages of the cell cycle indicate that changes in damage could be due solely to changes in chromatin geometry. But survival is not exclusively a function of damage.     

Phosphorylation of the lysine-rich histones throughout the cell cycle.

The phosphorylating of the lysine-rich histone at various stages in the cell cycle has been studied. In rapidly dividing cell populations the lysine-rich histone is phosphorylated rapidly after synthesis and more slowly once bound to the chromosome. The half-life of hydrolysis of such interphase phosphorylation in 5 hr except during mitosis when the phosphata hydrolysis increases almost three-fold. During mitosis there is extensive phosphorylation at sites different from those phosphorylated during interphase and a smaller measure of sites common to both mitotic and interphase cells. The sites of mitotic phosphorylation are most critically distinguished from those phosphorylated in interphase by the rapidly hydrolysis of M-phase phosphohistone when the cells divide and enter the G1 phase of the cell cycle.     

Microtubule stress modifies intra-nuclear location of Msh2 in mouse embryonic fibroblasts

The maintenance of genomic stability in mitotic and meiotic cycles through mismatch repair (MMR) demands the coordination of MMR functions with multiple processes including cell cycle traverse, linked changes in microtubule dynamics, protein translocation at chromatin sites and checkpoint activation. We have studied changes in the intracellular location of the MMR protein Msh2 in response to mitosis, microtubule disruption by colcemid and DNA damage induction by cis-platin in mouse embryonic fibroblasts (MEFs). Image analysis indicated that MEFs have a normally high nuclear retention of Msh2 during interphase with a precipitous dispersal of protein from chromatin sites into the cytoplasm at mitosis. Dispersal was also observed in cisplatin- and colcemid-treated interphase MEFs without any change in the overall Msh2 levels throughout the cell cycle. There was no evidence of co-localization of the punctate cytoplasmic Msh2 foci with any microtubule structures and knockout of Msh2 altered neither the extent of microtubule disruption nor the functional activation of the spindle assembly checkpoint by colcemid. Critically, extra-nuclear relocation of protein did not alter the ability to mount an Msh2-dependent G2 checkpoint delay in response to cisplatin-induced DNA damage. Depletion of the nuclear pool of Msh2 protein in cells undergoing dispersal was found to involve a rapid relocation of protein from AT-rich chromatin sites as defined by coassociation studies exploiting a newly-characterized base-pair preference of the fluorescent DNA binding probe DRAQ5. The study reveals the unexpected mobility of MMR protein pools during the MEF cell cycle and in response to different stress-inducing agents. The results link for the first time microtubule-integrity with intra-nuclear Msh2 protein dynamics. The high nuclear retention of Msh2 in interphase MEFs is in contrast to human tumor cells while the observations on protein dispersal suggest that only low levels of nuclear-located Msh2 are needed for G2 checkpoint activation by DNA damage.     

Microtubule Stress Modifies Intra-Nuclear Location of Msh2 in Mouse Embryonic Fibroblasts

The maintenance of genomic stability in mitotic and meiotic cycles through mismatch repair (MMR) demands the co-ordination of MMR functions with multiple processes including cell cycle traverse, linked changes in microtubule dynamics, protein translocation at chromatin sites and checkpoint activation. We have studied changes in the intracellular location of the MMR protein Msh2 in response to mitosis, microtubule disruption by colcemid and DNA damage induction by cis-platin in mouse embryonic fibroblasts (MEFs). Image analysis indicated that MEFs have a normally high nuclear retention of Msh2 during interphase with a precipitous dispersal of protein from chromatin sites into the cytoplasm at mitosis. Dispersal was also observed in cisplatin- and colcemid-treated interphase MEFs without any change in the overall Msh2 levels throughout the cell cycle. There was no evidence of co-localisation of the punctate cytoplasmic Msh2 foci with any microtubule structures and knockout of Msh2 altered neither the extent of microtubule disruption nor the functional activation of the spindle assembly checkpoint by colcemid. Critically, extra-nuclear relocation of protein did not alter the ability to mount an Msh2-dependent G2 checkpoint delay in response to cisplatin-induced DNA damage. Depletion of the nuclear pool of Msh2 protein in cells undergoing dispersal was found to involve a rapid relocation of protein from AT-rich chromatin sites as defined by co-association studies exploiting a newly-characterised base-pair preference of the fluorescent DNA binding probe DRAQ5. The study reveals the unexpected mobility of MMR protein pools during the MEF cell cycle and in response to different stress-inducing agents. The results link for the first time microtubule-integrity with intra-nuclear Msh2 protein dynamics. The high nuclear retention of Msh2 in interphase MEFs is in contrast to human tumour cells while the observations on protein dispersal suggest that only low levels of nuclear-located Msh2 are needed for G2 checkpoint activation by DNA damage.     

Colcemid inhibits the rejoining of the nucleotide excision repair of UVC-induced DNA damages in Chinese hamster ovary cells

In our previous study, we found that colcemid, an inhibitor of mitotic spindle, promotes UVC-induced apoptosis in Chinese hamster ovary cells (CHO.K1). In this study, a brief treatment of colcemid on cells after but not before UV irradiation could synergistically reduce the cell viability. Although colcemid did not affect the excision of UV-induced DNA damages such as [6-4] photoproducts or cyclobutane pyrimidine dimers, colcemid accumulated the DNA breaks when it was added to cells following UV-irradiation. This colcemid effect required nucleotide excision repair (NER) since the same accumulation of DNA breaks was barely or not detected in two NER defective strains of CHO cells, UV5 or UV24. Furthermore, the colcemid effect was not due to semi-conservative DNA replication or mitosis since the colcemid-caused accumulation of DNA breaks was also seen in non-replicating cells. Moreover, colcemid inhibited rejoining of DNA breaks accumulated by hydroxyurea/cytosine arabinoside following UV irradiation. Nevertheless, colcemid did not affect the unscheduled DNA synthesis as assayed by the incorporation of bromodeoxyuridine. Taken together, our results suggest that colcemid might inhibit the step of ligation of NER pathways.     

Colcemid inhibits the rejoining of the nucleotide excision repair of UVC-induced DNA damages in Chinese hamster ovary cells

In our previous study, we found that colcemid, an inhibitor of mitotic spindle, promotes UVC-induced apoptosis in Chinese hamster ovary cells (CHO.K1). In this study, a brief treatment of colcemid on cells after but not before UV irradiation could synergistically reduce the cell viability. Although colcemid did not affect the excision of UV-induced DNA damages such as [6–4] photoproducts or cyclobutane pyrimidine dimers, colcemid accumulated the DNA breaks when it was added to cells following UV-irradiation. This colcemid effect required nucleotide excision repair (NER) since the same accumulation of DNA breaks was barely or not detected in two NER defective strains of CHO cells, UV5 or UV24. Furthermore, the colcemid effect was not due to semi-conservative DNA replication or mitosis since the colcemid-caused accumulation of DNA breaks was also seen in non-replicating cells. Moreover, colcemid inhibited rejoining of DNA breaks accumulated by hydroxyurea/cytosine arabinoside following UV irradiation. Nevertheless, colcemid did not affect the unscheduled DNA synthesis as assayed by the incorporation of bromodeoxyuridine. Taken together, our results suggest that colcemid might inhibit the step of ligation of NER pathways.     

Spectrin redistributes to the cytosol and is phosphorylated during mitosis in cultured cells

Dramatic changes in morphology and extensive reorganization of membrane-associated actin filaments take place during mitosis in cultured cells, including rounding up; appearance of numerous actin filament-containing microvilli and filopodia on the cell surface; and disassembly of intercellular and cell-substratum adhesions. We have examined the distribution and solubility of the membrane-associated actin-binding protein, spectrin, during interphase and mitosis in cultured CHO and HeLa cells. Immunofluorescence staining of substrate-attached, well-spread interphase CHO cells reveals that spectrin is predominantly associated with both the dorsal and ventral plasma membranes and is also concentrated at the lateral margins of cells at regions of cell-cell contacts. In mitotic cells, staining for spectrin is predominantly in the cytoplasm with only faint staining at the plasma membrane on the cell body, and no discernible staining on the membranes of the microvilli and filopodia (retraction fibers) which protrude from the cell body. Biochemical analysis of spectrin solubility in Triton X-100 extracts indicates that only 10-15% of the spectrin is soluble in interphase CHO or HeLa cells growing attached to tissue culture plastic. In contrast, 60% of the spectrin is soluble in mitotic CHO and HeLa cells isolated by mechanical "shake-off" from nocodazole-arrested synchronized cultures, which represents a four- to sixfold increase in the proportion of soluble spectrin. This increase in soluble spectrin may be partly due to cell rounding and detachment during mitosis, since the amount of soluble spectrin in CHO or HeLa interphase cells detached from the culture dish by trypsin-EDTA or by growth in spinner culture is 30-38%. Furthermore, mitotic cells isolated from synchronized spinner cultures of HeLa S3 cells have only 2.5 times as much soluble spectrin (60%) as do synchronous interphase cells from these spinner cultures (25%). The beta subunit of spectrin is phosphorylated exclusively on serine residues both in interphase and mitosis. Comparison of steady-state phosphorylation levels of spectrin in mitotic and interphase cells demonstrates that solubilization of spectrin in mitosis is correlated with a modest increase in the level of phosphorylation of the spectrin beta subunit in CHO and HeLa cells (a 40% and 70% increase, respectively). Two-dimensional phosphopeptide mapping of CHO cell spectrin indicates that this is due to mitosis-specific phosphorylation of beta-spectrin at several new sites. This is independent of cell rounding and dissociation from other cells and the substratum, since no changes in spectrin phosphorylation take place when cells are detached from culture dishes with trypsin-EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA Damage-Induced Accumulation of Centrosomal Chk1 Contributes to its Checkpoint Function

The checkpoint kinase Chk1 is an established transducer of ATR- and ATM-dependent signalling in response to DNA damage. In addition to its nuclear localization, Chk1 localizes to interphase centrosomes and thereby negatively regulates entry into mitosis by preventing premature activation of cyclin B-Cdk1 during unperturbed cell cycles. Here, we demonstrate that DNA damage caused by ultraviolet irradiation or hydroxyurea treatment leads to centrosomal accumulation of endogenous Chk1 in normal human BJ fibroblasts and in ATR- or ATM-deficient fibroblasts. Chemical inhibition of ATR/ATM by caffeine led to enhanced centrosomal Chk1 deposition associated with nuclear Chk1 depletion. In contrast to normal or ATM-deficient fibroblasts, genetically ATR-deficient Seckel-fibroblasts showed detectable constitutive centrosomal accumulation of Chk1 even in the absence of exogenous insults. After DNA damage, the centrosomal fraction of Chk1 was found to be phosphorylated at ATR/ATM phosphorylation sites. Forced immobilization of kinase-inactive but not wild-type Chk1 to centrosomes resulted in a G2/M checkpoint defect. Finally, both DNA damage, and forced centrosomal expression of Chk1 in the absence of genotoxic treatments, induced centrosome amplification in a subset of cells, a phenomenon which could be suppressed by inhibition of ATM/ATR-mediated signaling. Taken together, our results suggest that accumulation of phosphorylated Chk1 at centrosomes constitutes an additional element in the DNA damage response. Centrosomal Chk1 induces G2/M cell cycle arrest and may evoke centrosome amplification, the latter possibly providing a backup mechanism for elimination of cells with impaired DNA damage checkpoints operating earlier during the cell cycle.     

UV-induced damage and repair in centromere DNA of yeast

Summary The centromere is the region within a chromosome that is required for proper segregation during mitosis and meiosis. Lesions in this sequence represent a unique type of damage, as loss of function could result in catastrophic loss of the genetic material of an entire chromosome. We have measured the induction by ultraviolet (UV) light of pyrimidine dimers in a 2550-bp restriction fragment that includes the centromere region of chromosome III in Saccharomyces cerevisiae. Yeast cells were exposed to ultraviolet light, cellular DNA was gently extracted, and subsequently treated with a UV-specific endonuclease to cleave all pyrimidine dimers. The sites of UV-specific nuclease scission within the centromere were determined by separating the DNA according to molecular weight, transferring the fragments to nitrocellulose, and hybridizing to a radiolabeled 624-bp fragment homologous to the centromere DNA from chromosome III. Several hotspots were identified in chromatin DNA from cells, as well as in irradiated deproteinized DNA. Double strand damage due to closely opposed pyrimidine dimers was also observed. At biological doses (35% survival) there are approximately 0.1 to 0.2 pyrimidine dimers per centromere. These dimers are efficiently repaired in the centromere and surrounding region.     

Cell cycle age dependence for radiation-induced G2 arrest: evidence for time-dependent repair

Exponentially growing eucaryotic cells, irradiated in interphase, are delayed in progression to mitosis chiefly by arrest in G2. The sensitivity of Chinese hamster ovary cells to G2-arrest induction by X rays increases through the cell cycle, up to the X-ray transition point (TP) in G2. This age response can be explained by cell cycle age-dependent changes in susceptibility of the target(s) for G2 arrest and/or by changes in capability for postirradiation recovery from G2-arrest damage. Discrimination between sensitivity changes and repair phenomena is possible only if the level of G2-arrest-causing damage sustained by a cell at the time of irradiation and the level ultimately expressed as arrest can be determined. The ability of caffeine to ameliorate radiation-induced G2 arrest, while inhibiting repair of G2-arrest-causing damage makes such an analysis possible. CHO cell monolayers were irradiated (1.5 Gy), then exposed to 5 mM caffeine for periods of 0-10 hr. Cell progression was monitored by the mitotic cell selection procedure. In the presence of caffeine, progression of irradiated cells was relatively unperturbed, but on caffeine removal, G2 arrest was expressed. The duration of G2 arrest was independent of the length of the prior caffeine exposure and, since cells of all ages were ultimately examined, the duration of arrest was also independent of cell cycle age at the time of irradiation. This finding indicates that the target for G2-arrest induction is present throughout the cell cycle and that the level of G2-arrest damage incurred is initially constant for all cell cycle phases. The data are consistent with the existence of a time-dependent recovery mechanism to explain the age dependence for radiation induction of G2 arrest.     

Negative regulation of condensin I by CK2-mediated phosphorylation

Condensin I, which plays an essential role in mitotic chromosome assembly and segregation in vivo, constrains positive supercoils into DNA in the presence of adenosine triphosphate in vitro. Condensin I is constitutively present in a phosphorylated form throughout the HeLa cell cycle, but the sites at which it is phosphorylated in interphase cells differ from those recognized by Cdc2 during mitosis. Immunodepletion, in vitro phosphorylation, and immunoblot analysis using a phospho-specific antibody suggested that the CK2 kinase is likely to be responsible for phosphorylation of condensin I during interphase. In contrast to the slight stimulatory effect of Cdc2-induced phosphorylation of condensin I on supercoiling, phosphorylation by CK2 reduced the supercoiling activity of condensin I. CK2-mediated phosphorylation of condensin I is spatially and temporally regulated in a manner different to that of Cdc2-mediated phosphorylation: CK2-dependent phosphorylation increases during interphase and decreases on chromosomes during mitosis. These findings are the first to demonstrate a negative regulatory mode for condensin I, a process that may influence chromatin structure during interphase and mitosis.     

Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA

Using a transient gene expression assay to measure host cell reactivation, the effects of cyclobutane dimer and noncyclobutane dimer uv photoproducts on expression of a reporter gene were examined in normal and repair-deficient Chinese hamster ovary (CHO) cell lines. Ultraviolet damage in plasmid pRSV beta gal DNA, containing the Escherichia coli beta-galactosidase gene, resulted in reduced reporter gene expression in both uv-hypersensitive mutant CHO cell lines UV5 and UV61 relative to wild-type, parental AA8 cells. However, the effects of uv irradiation of transfected plasmid DNA on gene activity were reduced in UV61, a mutant with normal (6-4) photoproduct repair, compared to UV5, which is deficient in (6-4) photoproduct repair; this reduction correlated with the intermediate uv-hypersensitivity of UV61. Selective removal of cyclobutane dimers by in vitro photoreactivation of uv-irradiated plasmid DNA prior to transfection substantially increased reporter gene activity in both uv-hypersensitive mutant cell lines. This increase was significantly greater in UV61 than in UV5, consistent with UV5 being deficient in repair of both (6-4) photoproducts and cyclobutane dimers. These results suggest that unrepaired (6-4) photoproducts in transfected pRSV beta gal plasmid DNA are responsible for a significant fraction of the reduction in transient gene expression observed in recipient uv-hypersensitive CHO cell mutants.     

Condensed chromatin and cell inactivation by single-hit kinetics

Mammalian cells are extremely sensitive to gamma rays at mitosis, the time at which their chromatin is maximally condensed. The radiation-induced killing of mitotic cells is well described by single-hit inactivation kinetics. To investigate if radiation hypersensitivity by single-hit inactivation correlated with chromatin condensation, Chinese hamster ovary (CHO) K1 (wild-type) and xrs-5 (radiosensitive mutant) cells were synchronized by mitotic shake-off procedures and the densities of their chromatin cross sections and their radiosensitivities were measured immediately and 2 h into G1 phase. The chromatin of G1-phase CHO K1 cells was dispersed uniformly throughout their nuclei, and its average density was at least three times less than in the chromosomes of mitotic CHO K1 cells. The alpha-inactivation co-efficient of mitotic CHO K1 cells was approximately 2.0 Gy(-1) and decreased approximately 10-fold when cells entered G1 phase. The density of chromatin in CHO xrs-5 cell chromosomes at mitosis was greater than in CHO K1 cell chromosomes, and the radiosensitivity of mitotic CHO xrs-5 cells was the greatest with alpha = 5.1 Gy(-1). In G1 phase, CHO xrs-5 cells were slightly more resistant to radiation than when in mitosis, but a significant proportion of their chromatin was found to remain in condensed form adjacent to the nuclear membrane. These studies indicate that in addition to their known defects in DNA repair and V(D)J recombination, CHO xrs-5 cells may also be defective in some process associated with the condensation and/or dispersion of chromatin at mitosis. Their radiation hypersensitivity could result, in part, from their DNA remaining in compacted form during interphase. The condensation status of DNA in other mammalian cells could define their intrinsic radiosensitivity by single-hit inactivation, the mechanism of cell killing which dominates at the dose fraction size (1.8-2.0 Gy) most commonly used in radiotherapy.     

The alga Chlamydomonas reinhardtii UVS11 gene is responsible for cell division delay and temporal decrease in histone H1 kinase activity caused by UV irradiation

The aim of the present work was to study the possible role of the UVS11 gene of the alga Chlamydomonas reinhardtii, in regulation of the cell cycle. To characterize the defect of a uvs11 mutant in respect to DNA damage-dependent cell cycle arrest, we examined first the influence of the tubulin-destabilizing drug methyl benzimidazole-2-yl-carbamate (MBC) on inhibition of mitosis in response to UV 254nm. Then the growth and reproductive processes and activity of cyclin-dependent kinases (CDK)-like kinases during the cell cycle of C. reinhardtii were investigated. In both, the wild type and the uvs11 mutant strain were compared under standard conditions and after DNA damage caused by UV 254nm. We assume the green alga C. reinhardtii possesses control mechanisms allowing to stop the cell cycle progression before mitosis in response to DNA damage. The results indicate that the uvs11 mutant is not able to stop the cell cycle after UV irradiation. We suggest that a product of the UVS11 gene affects cell response to DNA damage and influences a decrease in histone H1 kinase activity.     

Possible mechanisms of the occurrence of chromosome aberrations. I. Patterns in the occurrence of aberrations induced by ultraviolet irradiation

The frequency of chromosome aberrations induced by UV light at wavelengths 254, 265, 280 and 302 using doses 2-10 J/m2 in the primary culture of mouse embryonic fibroblasts during the G1, S and G2 phases was studied at metaphase of the first mitosis. Two classes of chromosome aberrations were distinguished. These classes differ in the time intervals of the final establishment of the cell cycle. The aberrations of the class 1 emerge before the beginning of prometaphase (possibly, at interphase). Formation of the second class aberrations is completed during the metaphase. It is shown that the class 1 aberrations occur with almost the same rate in approx. 7% of cells, irrespective of the cell cycle, irradiation dose and wavelength. It is suggested that these aberrations arise as a result of indirect UV action on the chromosome structures; the mechanism of their emergence does not depend on DNA replication. The class 2 aberrations do not appear after UV irradiation during the post-DNA-synthetic G2 phase of the cell cycle. However, after UV treatment at the G1 or S periods, they represent the majority of aberrations and their rate increases almost monotonously with the radiation dose. The UV action spectrum for these aberrations coincides with the adsorption spectrum of thymidine and the action spectrum for DNA cross-links. Thus, it may be inferred that formation of DNA cross-links following thymine dimerization is the first step in formation of UV-induced aberrations of the class 2. The passage of cells through DNA replication is a very important step in the process of their emergence.     

Polo-like kinase 1 inhibits DNA damage response during mitosis

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.     

Polo-like kinase 1 inhibits DNA damage response during mitosis

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.     

Loss of thymine dimers from mammalian cell DNA. The kinetics for antibody-binding sites are not the same as that for T4 endonuclease V sites

Antiserum specific for thymine-containing dimers was used to assay DNA isolated from ultraviolet-irradiated cells following different repair periods. A 50% loss in antibody-binding sites was evident 1 h post-irradiation, and within 4 h 80% of the sites were removed. This result contrasts with data obtained with dimer-specific T4 endonuclease V and does not appear to be due to masking of the dimers by repair enzymes. T4 endonuclease V treatment of ultraviolet-irradiated DNA at 0 degree C resulted in conversion of the thymine dimers to apyrimidinic sites. This did not result in loss of antigenicity in either PM2 or CHO cell DNA. Likewise, treatment of ultraviolet-irradiated CHO cell DNA with T4 endonuclease at 37 degrees C did not change its antigenicity. These results suggest that aglycosylation of the dimers is not responsible for their inability to bind dimer-specific antibody 2-4 h post-irradiation. The possibility that T4 endonuclease V and the antiserum have different specificities for different dimers is discussed.