Role of DNA damage in the formation of radiation damage to chromosomes

The data are reviewed on the role of various DNA lesions in the formation of structural damages to chromosomes. The concepts are developed that the molecular damages to nuclear DNA induce chromosome mutagenesis.     

DNA damage caused by ionizing radiation.

A survey is given of continuous-time Markov chain models for ionizing radiation damage to the genome of mammalian cells. In such models, immediate damage induced by the radiation is regarded as a batch-Poisson arrival process of DNA double-strand breaks (DSBs). Enzymatic modification of the immediate damage is modeled as a Markov process similar to those described by the master equation of stochastic chemical kinetics. An illustrative example is the restitution/complete-exchange model. The model postulates that, after being induced by radiation, DSBs subsequently either undergo enzymatically mediated restitution (repair) or participate pairwise in chromosome exchanges. Some of the exchanges make irremediable lesions such as dicentric chromosome aberrations. One may have rapid irradiation followed by enzymatic DSB processing or have prolonged irradiation with both DSB arrival and enzymatic DSB processing continuing throughout the irradiation period. Methods for analyzing the Markov chains include using an approximate model for expected values, the discrete-time Markov chain embedded at transitions, partial differential equations for generating functions, normal perturbation theory, singular perturbation theory with scaling, numerical computations, and certain matrix methods that combine Perron-Frobenius theory with variational estimates. Applications to experimental results on expected values, variances, and statistical distributions of DNA lesions are briefly outlined. Continuous-time Markov chains are the most systematic of those radiation damage models that treat DSB-DSB interactions within the cell nucleus as homogeneous (e.g., ignore diffusion limitations). They contain virtually all other relevant homogeneous models and semiempirical summaries as special cases, limiting cases, or approximations. However, the Markov models do not seem to be well suited for studying spatial dependence of DSB interactions, which is known to be important in some situations.     

Characterization of radiation damage to DNA by reaction with borohydride.

Irradiation of aqueous solutions of native calf thymus DNA with x-rays produced functional groups that reacted with sodium borohydride. The DNA was labeled with tritium from NaB3H4 to the extent of 2.0 x 10(-10) atom/dalton/rad. The presence of cysteamine or other radical scavengers, or saturation of the solution with nitrogen during irradiation decreased the labeling. After mild acid hydrolysis, the major tritium-containing moiety was identical with 2,3-dihydroxy-2-methylpropanoic acid in all chromatographic systems tested. The suggested mechanism of labeling involved reduction by borohydride of the potential aldehyde at carbon 6 of thymine glycol residues present in the irradiated DNA.     

DNA damage in cells exposed to ionizing radiation

Ionizing radiation induces variety of structural lesions in DNA of irradiated organisms. Their formation depends largely on the degree of cell oxygenation, the level of endogenous antioxidants, on DNA-protein complexes and compactization of DNA in the chromatin and activity of DNA repair systems. All ionizing radiation-induced DNA lesions can arbitrarily be divided into two groups. Group 1 includes singly damaged sites (single-sites): base modification, single-strand breaks, alkaline-labile sites (including a basic sites). Group 2 contains: locally multiply damaged sites (clustered lesions), double-strand breaks, intermolecular cross-links. The yields of lesions of group 2 increases with high linear energy transfer of radiation and these lesions play a dominant role in the radiation death, formation of chromosome and gene mutations, cell transformation.     

DNA放射损伤与p53

电离辐射等多种因素可以引起DNA损伤,表现为碱基改变、DNA双链断裂(DNA double-strand breaks,DSBs)和DNA单链断裂(Single-strand breaks,SSBs)等多种形式。DNA损伤后,细胞发生应答,即引起细胞周期阻滞和/或细胞程序性死亡,以减少损伤引起的染色体畸变和基因组不稳定。在细胞应答过程中,p53蛋白水平和活性均发生变化,介导细胞周期阻滞、程序性死亡,并直接参与DNA损伤修复过程。     

Ionizing radiation damage to DNA: molecular aspects

Radioresistant tumor cells are found in tumor specimens from patients in whom radiotherapy has failed or whose tumors have recurred after therapy. This suggests that inherent cellular radioresistance may in part underlie the failure of radiotherapy, and therefore determination of the presence of resistant cells within a tumor might be a useful predictor of response to radiation therapy. Most standard clonogenic assays of radiation response are time-consuming, and alternative assays of radiation response are being sought. In an earlier publication (J. L. Schwartz et al., Int. J. Radiat. Oncol. Biol. Phys. 15, 907-912, 1988), we reported that radioresistant human tumor cells rejoin DNA double-strand breaks, as measured by DNA neutral filter elution (pH 9.6), faster than more sensitive cell lines. To determine whether DNA elution might have potential as a rapid predictive assay, we examined the relationship between the rate of DNA double-strand break rejoining and radiosensitivity in nine first-passage-after-explant squamous cell carcinomas under conditions that minimized the influence of nontumor and nonclonogenic cells. The frequency of DNA double-strand breaks measured 1 h after irradiation with 100 Gy 60Co gamma rays was used as an estimate of relative rejoining rate. This number is a reflection of both the initial DNA double-strand break frequency and the amount of repair that occurs in 1 h. The relative break frequency was compared to radiosensitivity as measured by standard clonogenic survival assays in later passages (p3-p14) of these same cells. A significant relationship (r = 0.61, P less than 0.01) was found between break frequency measured in first-passage cells and radiosensitivity measured in later passages, suggesting that the neutral elution assay as described here has some promise as a relatively rapid assay of the radiosensitivity of human tumor cells.     

Bleomycin-iron complexes and DNA radiation damage.

Radiation in the form of high-energy electrons dose-dependently activates bleomycin-Fe3+ in oxygen-containing DNA solutions. This activation causes a DNA fragmentation and a release of oxidative degradation products from the DNA. During irradiation, bleomycin-chelated Fe3+ is reduced to Fe2+. The kinetics of DNA base propenal-formation (measured by reaction with thiobarbituric acid) and iron-reduction (measured by bathophenanthroline chelation) are similar, with a yield of 1 mol base propenal/6 mol Fe3+ reduced. The activation of bleomycin-Fe3+ by irradiation could be instrumental in the synergistic action of radiotherapy and bleomycin observed on simultaneous administration in vivo.     

Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging

Ionizing radiation (IR) is a common mode of cancer therapy, where DNA damage is the major reason of cell death. Here, we use an assay based on fluorescence imaging of single damaged DNA molecules isolated from radiated lymphocytes, to quantify IR induced DNA damage. The assay uses a cocktail of DNA-repair enzymes that recognizes and excises DNA lesions and then a polymerase and a ligase incorporate fluorescent nucleotides at the damage sites, resulting in a fluorescent “spot” at each site. The individual fluorescent spots can then be counted along single stretched DNA molecules and the global level of DNA damage can be quantified. Our results demonstrate that inclusion of the human apurinic/apyrimidinic endonuclease 1 (APE1) in the enzyme cocktail increases the sensitivity of the assay for detection of IR induced damage significantly. This optimized assay also allowed detection of a cooperative increase in DNA damage when IR was combined with mild hyperthermia, which is sometimes used as an adjuvant in IR therapy. Finally, we discuss how the method may be used to identify patients that are sensitive to IR and other types of DNA damaging agents.     

Structural damage to meiotic chromosomes impairs DNA recombination and checkpoint control in mammalian oocytes

Meiosis in human oocytes is a highly error-prone process with profound effects on germ cell and embryo development. The synaptonemal complex protein 3 (SYCP3) transiently supports the structural organization of the meiotic chromosome axis. Offspring derived from murine Sycp3^−/− females die in utero as a result of aneuploidy. We studied the nature of the proximal chromosomal defects that give rise to aneuploidy in Sycp3^−/− oocytes and how these errors evade meiotic quality control mechanisms. We show that DNA double-stranded breaks are inefficiently repaired in Sycp3^−/− oocytes, thereby generating a temporal spectrum of recombination errors. This is indicated by a strong residual γH2AX labeling retained at late meiotic stages in mutant oocytes and an increased persistence of recombination-related proteins associated with meiotic chromosomes. Although a majority of the mutant oocytes are rapidly eliminated at early postnatal development, a subset with a small number of unfinished crossovers evades the DNA damage checkpoint, resulting in the formation of aneuploid gametes.     

Modeling radiation damage to DNA using radionuclides incorporated into polynucleotides

Because of a great variety and different reparability of radiation-induced DNA lesions it is difficult to evaluate the radiobiological significance of certain individual alterations. It is suggested that the radionuclides incorporated into DNA can be used to imitate different types of radiation damages to DNA. Both qualitative and quantitative aspects of the problem are discussed.     

Eukaryotic DNA metabolism : Are deoxyribonucleotides channeled to replication sites?

DNA precursor biosynthesis is closely coordinated with DNA replication itself. In prokaryotic systems, firm evidence supports the idea that this coordination is achieved through the action of multienzyme complexes that physically link the synthesis of deoxyribonucleotides with their utilization in DNA replication. Much evidence favors a similar channeling mechanism in eukaryotes. However, recent studies suggest strongly that in mammalian cells DNA precursors are synthesized in cytoplasm and are then transported into the nucleus. This article reviews the pertinent evidence, attempts to reconcile contradictory findings, and highlights areas that need further investigation.