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S M Chiu

Publications and source records attributed to S M Chiu.

35 records · Page 2Linked to original sources
PubMed1987

Yield of DNA-protein cross-links in gamma-irradiated Chinese hamster cells.

gamma-Irradiation of Chinese hamster V79 cells increases the percentage of nuclear DNA cross-linked to proteins. Studies were carried out to ascertain whether the radiation-induced increase in DNA-protein cross-links (DPC) is due to an increase in the number of DNA fragments which are cross-linked to protein or to an increase in the size of bound DNA fragments. Cells were prelabeled with [3H]thymidine and irradiated (10-600 Gy), and DPCs were collected on nitrocellulose filters. Native gel analyses of the DNA recovered from the filters indicate that the number average molecular weight of cross-linked DNA (1.22 X 10(7) Da) is the same in unirradiated cells and in cells given up to 100 Gy. Assuming 5 pg of DNA per V79 cell, it was possible to calculate that there are approximately 6 X 10(3) DPC per unirradiated cell and that 150 DPC are formed per gray of gamma-radiation for doses of 0-100 Gy. Thus, radiation increases the number of new linkages between DNA and protein. At radiation doses greater than 200 Gy the percentage of nuclear DNA cross-linked to protein approaches a plateau value. The number of DPC (greater than 6 X 10(4)) formed at higher doses is within the range of the estimated number of DNA attachment sites on the nuclear matrix.

Animals↗
PubMed1987

Induction of DNA strand breaks in transcriptionally active DNA sequences of mouse cells by low doses of ionizing radiation.

The efficiency of DNA single-strand break induction was measured in transcriptionally active DNA, transcriptionally inert satellite DNA, and bulk DNA sequences of mouse L929 cells using the alkaline filter elution assay. The cells were exposed to increasing doses of X-radiation up to 1000 rad. DNA which either eluted from or was retained on polycarbonate filters during the assays was collected onto nitrocellulose filters and hybridized against radiolabeled poly(A+)RNA (to probe transcribing DNA sequences) or mouse satellite DNA. The increasing rate and extent of elution of bulk DNA or specific DNA sequences after increasing radiation doses was taken as a measure of the increased frequency of radiation-induced DNA strand breaks. The results indicate that a significant fraction of transcriptionally active DNA contains endogenous strand breaks. With increasing dose, the efficiency of radiation-induced DNA strand breakage in bulk, transcriptionally active and satellite DNA sequences was observed to be the same when the sum of all eluted DNA was considered. However, the early eluting fractions contained DNA which was enriched in active sequences. Since DNA elutes as a function of size, the early fractions contain smaller DNA than later fractions. Therefore, our results indicate that the fraction of active sequences which elutes early resides on smaller fragments on the average than the later eluting DNA, and that even low doses of radiation preferentially cause breaks in regions of DNA containing active sequences.

Animals↗
PubMed1987

The formation, identification, and significance of DNA-protein cross-links in mammalian cells.

DNA-protein cross-links (DPC) are formed by a variety of radiations and chemicals which act via free radical formation. Covalency is inferred from the resistance of the cross-links to harsh treatments. In mammalian cells, a background of DPC (6000 per V79 cell) may result from normal associations of chromosomal loops with the nuclear protein matrix. After ionizing radiation, the elevated level of DPC (150 per Gy per V79 cell) are enriched in actively transcribing DNA and in a subset of proteins of the nuclear matrix. DPC formation is reduced by hydroxyl radical scavengers, by oxygen, and by hypertonic medium and is enhanced by hypotonic medium and by removal of intracellular glutathione. DPC are repaired more slowly than single-strand breaks and not at all when formed during metaphase. During the postirradiation period, changes in the sequence composition of the DNA of residual DPC are consistent with the preferential repair of DPC in actively expressed genes. Excision repair mechanisms have been proposed. Unrepaired DPC may block normal functions of the nuclear matrix, such as replication and transcription.

Animals↗
PubMed1986

Modification of DNA damage in transcriptionally active vs. bulk chromatin.

Our previous experiments have demonstrated that regions of nuclear chromatin, containing transcriptionally active DNA sequences and associated with the nuclear matrix, are hypersensitive to the production of both single-strand breaks and DNA-protein cross-links upon gamma-irradiation of exponentially growing mammalian cells. In this study, we have irradiated Chinese hamster V79 cells in buffered saline with or without DMSO to scavenge hydroxyl radicals and in buffered salines of various tonicities to expand or condense chromatin. The yield of DNA-protein cross-links was assayed by a nitrocellulose filter binding technique and the DNA recovered from the cross-links hybridized to 125I-poly(A+)RNA to determine the relative frequency of transcriptionally active sequences in the cross-links compared to the bulk DNA. In all cases, the data show that active DNA is affected to a greater extent than bulk, primarily inactive DNA. The more extensive alteration of the level of ionizing radiation-induced damage in active DNA by the diffusible agents tested suggests that other agents, such as chemical sensitizers and protectors, which need to diffuse to the nuclear DNA, may also be acting primarily on active, matrix-associated DNA.

Animals↗
PubMed1986

Nuclear matrix proteins are crosslinked to transcriptionally active gene sequences by ionizing radiation.

Unirradiated, exponentially growing Chinese hamster cells contain a low level (less than 5%) of their DNA firmly bound to protein, as measured by a filter-binding assay. That fraction of DNA is highly enriched in sequences which hybridize to poly(A+)RNA or ribosomal RNA. After 60 Gy gamma irradiation, the additional crosslinked DNA is also enriched in transcriptionally active sequences compared to bulk DNA, while DNA crosslinked by uv radiation has a frequency of active sequences which is no higher than the bulk DNA. DNA crosslinked to protein by gamma radiation but not by uv is largely released during a 4-h postirradiation incubation. The DNA which remains bound to protein during that period becomes depleted in active sequences; this is followed by an apparent restoration of the active gene-enriched protein complex found in unirradiated cells. When nuclear matrix-associated DNA was isolated free of the majority ("loop") DNA, an enrichment for active DNA sequences was found in the matrix-associated DNA, and the frequency of DNA-protein crosslinks was found to be 10- to 16-fold greater in the matrix fraction. Gel electrophoretic analysis of the crosslinking proteins identifies them as subset of proteins of the nuclear matrix. These data are consistent with known properties of the nuclear matrix and suggest that chromatin structure plays an important role in the formation and repair of gamma-radiation-induced DNA lesions.

Animals↗
PubMed1984

Differential processing of ultraviolet or ionizing radiation-induced DNA-protein cross-links in Chinese hamster cells.

The yield and repairability of DNA-protein cross-links have been compared after gamma- or U.V.-irradiation of Chinese hamster V79-379 lung fibroblasts. Using a filter-binding assay, cross-linked DNA can be specifically isolated after doses between 10 and 100 Gy of gamma-radiation and fluences between 20 and 300 J/m2 of U.V.-radiation. After ionizing radiation, the majority of DNA cross-linked to protein is released with biphasic kinetics, requiring 1 h for removal of 50 per cent of the cross-linked DNA and 24 h for 90 per cent release. In these cells, U.V.-induced cross-linked DNA is not removed; on the contrary, the yield of apparent DNA-protein complexes increases during postirradiation incubation. Prior gamma-irradiation, to initiate the associated repair system, does not stimulate release of U.V.-induced cross-linked DNA. Inhibition of protein synthesis by cycloheximide affects neither the removal of gamma-ray-induced cross-linked DNA nor the increase in U.V.-induced cross-linked DNA. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, slows the second phase of release after gamma-irradiation as well as the increase in apparent cross-links after U.V.-irradiation. Thus, even though both types of DNA-protein cross-links can be detected by the same assay, their structures or other factors must be substantially different, since the repair system for one type does not recognize the other.

Animals↗
PubMed1984

Gamma radiation as a probe of chromatin structure: damage to and repair of active chromatin in the metaphase chromosome.

Cobalt-60 gamma radiation has been employed as a means of preferentially damaging actively transcribing chromatin within interphase and metaphase Chinese hamster V79-379 lung fibroblasts. The single-strand size distribution and break frequency of bulk 3H-labeled DNA have been compared to those same parameters for active sequences, i.e., sequences complementary to 125I-labeled poly(A+)RNA. The results show that (a) sequences active during interphase are more sensitive than inactive sequences to single-strand break formation by gamma radiation even when the chromatin is condensed in metaphase, (b) repair of strand breaks in the bulk DNA is slower in metaphase than in interphase cells, but (c) during metaphase, repair is faster in active sequences than in the bulk DNA. Furthermore, this study demonstrates that chromatin structure can be probed within intact cells by a method which circumvents isolation of nuclei or chromatin and the use of exogenous nucleases.

Animals↗
PubMed1982

Hypersensitivity of DNA in transcriptionally active chromatin to ionizing radiation.

We have examined the size distribution of single-strand fragments of total 3H-labeled DNA and of DNA sequences complementary to specific probes in gamma-irradiated and unirradiated mouse L929 cells. Those DNA sequences which hybridize to rDNA or to poly(A+)RNA have lower number average molecular weights and sustain 5--6-times the number of single-strand breaks as do satellite DNA sequences or the bulk DNA. We therefore conclude that transcriptionally active DNA sequences are more susceptible to ionizing radiation-induced damage than are inactive sequences, and suggest that these differential susceptibilities are a likely consequence of differences in their chromatin organization.

Animals↗
PubMed1976

Genetic recombination of Coprinus. V. Repair synthesis of deoxyribonucleic acid and its relation to meiotic recombination.

Repair synthesis of DNA per se at pachytene is not needed for commitment to meiotic recombination although it is a necessary event to follow. Recombination frequency is governed by the rate of nicking and the time in which unrepaired nicks are allowed to match and crossover. Cold treatment at pachytene prevented repair synthesis, hence open nicks were accumulated to match and crossover, and a 3-fold increase in recombination resulted. The kinetics of cold temperature effect followed a quadratic function as shown by a computer simulation which agreed with our experimental data (Lu, 1974b). High temperature did not change the rate of repair synthesis. It did cause an increased nicking which led to a twofold increase in recombination and which entailed a higher rate of recovery repair synthesis.

Agaricales↗