Repair of HZE-particle-induced DNA double-strand breaks in normal human fibroblasts.
ABSTRACT DNA damage generated by high-energy and high-Z (HZE) particles is more skewed toward multiply damaged sites or clustered DNA damage than damage induced by low-linear energy transfer (LET) X and gamma rays. Clustered DNA damage includes abasic sites, base damages and single- (SSBs) and double-strand breaks (DSBs). This complex DNA damage is difficult to repair and may require coordinated recruitment of multiple DNA repair factors. As a consequence of the production of irreparable clustered lesions, a greater biological effectiveness is observed for HZE-particle radiation than for low-LET radiation. To understand how the inability of cells to rejoin DSBs contributes to the greater biological effectiveness of HZE particles, the kinetics of DSB rejoining and cell survival after exposure of normal human skin fibroblasts to a spectrum of HZE particles was examined. Using gamma-H2AX as a surrogate marker for DSB formation and rejoining, the ability of cells to rejoin DSBs was found to decrease with increasing Z; specifically, iron-ion-induced DSBs were repaired at a rate similar to those induced by silicon ions, oxygen ions and gamma radiation, but a larger fraction of iron-ion-induced damage was irreparable. Furthermore, both DNA-PKcs (DSB repair factor) and 53BP1 (DSB sensing protein) co-localized with gamma-H2AX along the track of dense ionization produced by iron and silicon ions and their focus dissolution kinetics was similar to that of gamma-H2AX. Spatial co-localization analysis showed that unlike gamma-H2AX and 53BP1, phosphorylated DNA-PKcs was localized only at very specific regions, presumably representing the sites of DSBs within the tracks. Examination of cell survival by clonogenic assay indicated that cell killing was greater for iron ions than for silicon and oxygen ions and gamma rays. Collectively, these data demonstrate that the inability of cells to rejoin DSBs within clustered DNA lesions likely contributes to the greater biological effectiveness of HZE particles.
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ABSTRACT: Bursts of free radicals produced by ionization of water in close vicinity to DNA can produce clusters of opposed DNA lesions and these are termed multiply damaged sites (MDS). How MDS are processed by the Escherichia coli DNA glycosylases, endonuclease (endo) III and endo VIII, which recognize oxidized pyrimidines, is the subject of this study. Oligonucleotide substrates were constructed containing a site of pyrimidine damage or an abasic (AP) site in close proximity to a single nucleotide gap, which simulates a free radical-induced single-strand break. The gap was placed in the opposite strand 1, 3 or 6 nt 5' or 3' of the AP site or base lesion. Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand break, although cleavage at position one 5' or 3' was reduced compared with cleavage at positions three or six 5' or 3'. Neither endo III nor endo VIII was able to remove the base lesion when the gap was positioned 1 nt 5' or 3' in the opposite strand. Cleavage of the modified pyrimidine by endo III increased as the distance increased between the base lesion and the opposed strand break. With endo VIII, however, DNA breakage at the site of the base lesion was equivalent to or less when the gap was positioned 6 nt 3' of the lesion than when the gap was 3 nt 3' of the lesion. Gel mobility shift analysis of the binding of endo VIII to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII. If the strand break in the MDS was replaced by an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed. These data show that processing of oxidized pyrimidines by endos III and VIII was strongly influenced by the position and type of lesion in the opposite strand, which could have a significant effect on the biological outcome of the MDS lesion.Nucleic Acids Research 03/1998; 26(4):932-41. · 8.28 Impact Factor
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ABSTRACT: Studies of ionizing radiations of different quality are discussed with particular emphasis on damage to DNA of mammalian cells. Three related themes are followed. Firstly, inactivation and mutation experiments with ultrasoft X-rays and slow heavy ions, coupled with theoretical analyses of the structures of the radiation tracks, have emphasized the biological importance of localized track features over nanometre dimensions. This led to the suggestion that the critical physical features of the tracks are the stochastic clusterings of ionizations, directly in or very near to DNA, resulting in clustered initial molecular damage including various combinations of breaks, base damages, cross-links, etc. in the DNA. The quantitative hypotheses imply that final cellular effects from high-LET radiations are dominated by their more severe, and therefore less repairable, clustered damage, and that these are qualitatively different from the dominant low-LET damage. Second, relative effectiveness of different types of radiation led to questions on the mechanisms of induction of chromosome exchanges. The high efficiency of ultrasoft X-rays, despite their very short track lengths, suggested that single sites of DNA damage may lead to exchanges by a molecular process involving interaction with undamaged DNA. Also it is shown that a single site-specific DNA break, introduced by restriction enzymes, sometimes leads to a large deletion when misrepaired by cell extracts. These deletions occur between short DNA repeats, and are therefore a form of 'illegitimate' recombination, but clearly do not involve the interaction of two damage sites. Third, it was shown that cells from patients with the radiosensitive disorder ataxia-telangiectasia (AT) lack a post-irradiation recovery process. The sensitivity of AT cells to high LET radiations was found to be reduced relative to that for normal cells, reinforcing the concept that high LET damage is less easy to repair. AT patients are prone to lymphoreticular cancers, and their cells show characteristic chromosomal rearrangements, which may be associated with misrepair at specific genomic sequences. Similarly, studies of radiation-induced leukaemia in the mouse have implicated rearrangement at specific interstitial chromosome sites, which are rich in telomere-like repeat sequences.International Journal of Radiation Biology 06/1993; 63(5):543-56. · 1.90 Impact Factor
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ABSTRACT: The induction of intracellular DNA strand breaks by X rays and various heavy charged particles was measured by the alkaline unwinding and alkaline and neutral filter elution techniques. No variations in strand break induction were found between the different cell lines under investigation. For a given particle, both the LET and the particle energy determined the efficiency to induce DNA lesions. RBE values for the total amount of induced strand breaks were always less than 1. For DNA double-strand breaks (DSBs), RBE values only slightly greater than 1 were determined for particle radiation with an LET around 300 keV/microns. Intracellular DSB/SSB ratios were found to be equivalent to data reported for in vitro systems using radioprotective conditions [Christensen et al., Int. J. Radiat. Biol. 22, 457-477, 1972; Taucher-Scholz et al., Adv. Space Res. 12(2-3), (2)73-(2)80, 1992]. Strand break rejoining as an indicator of cellular repair processes was detected even after high-LET irradiation (LET < or = 10,000 keV/microns). However, both the half-times of rejoining and the fraction of residual DNA breaks increased with the atomic number of the particle. After particle irradiation with LET values beyond 10,000 keV/microns, no rejoining of DNA strand breaks was found.Radiation Research 07/1993; 135(1):46-55. · 2.70 Impact Factor