The role of DNA repair processes in the repair of cytogenetic disorders. The participation of slow to repair DNA damage in the repair of cytogenetic disorders

Caffeine was used to study the kinetics of cytogenetic damages repair in Chinese hamster fibroblasts. Its half-time (90 min) was shown to correlate with that of repair of slowly repaired DNA damages. The caffeine-induced increase in the number of irreparable DNA damages, attributed to inhibition of double-strand break repair, is in a quantitative correlation with the effect of the cytogenetic damage modification.     

Delayed repair of DNA single-strand breaks does not increase cytogenetic damage

DNA damage and cytogenetic effects of ionizing radiation were investigated in Chinese hamster ovary (CHO) cells and unstimulated human peripheral blood lymphocytes. DNA damage and repair were analysed by alkaline elution under conditions that predominantly measured DNA single-strand breaks (ssb). X-radiation (2.5 Gy) induced ssb in both CHO cells and unstimulated lymphocytes, and the breaks were repaired within 30 and 90 min, respectively. This rapid repair was delayed by the poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide (3AB). The cytogenetic effects of the 3AB-induced delay in DNA repair were examined by analysing sister chromatid exchange (SCE) frequency in CHO cells and fragmentation of prematurely condensed chromosomes (PCC) in unstimulated human lymphocytes after 2.5 Gy of X-rays. Although 3AB delayed the rejoining of DNA ssb, this delay did not result in increased cytogenetic damage manifested as either SCE or fragmentation of PCC. These results indicate that the rapidly rejoining DNA ssb are not important in the production of chromosome damage.     

Mechanisms of DNA repair disorders in human cells. Genome instability in lymphocytes of patients with myopathy related to DNA repair disorders

Increased level of cytogenetic damages was observed in lymphocytes of 10 patients with muscular dystrophy (Duchenne, Erba, Landuzi--Degerin). Analysis of DNA repair synthesis revealed inhibition of this process in lymphocytes of 10 patients observed. On the other hand, decreased reactivation of vaccinia virus and increased level of virus mutagenesis induced by 4-nitroquinoline-1-oxide and bleomycin was noted in the experiments with lymphocytes of 3 patients observed.     

Evaluating the effects of genetic variants of DNA repair genes using cytogenetic mutagen sensitivity approaches

Mutagen sensitivity, measured in short-term cultures of peripheral blood lymphocytes by cytogenetic endpoints, is an indirect measure for DNA repair capacity and has been used for many years as a biomarker for intrinsic susceptibility for cancer. In this article, we briefly give an overview of the different cytogenetic mutagen sensitivity approaches that have been used successfully to evaluate the biological effects of polymorphisms in DNA repair genes based on a current review of the literature and based on the need for biomarkers that would allow the characterization of the biological and functional significance of such polymorphisms. We also address some of the future challenges facing this emerging area of research.     

DNA repair/pro-apoptotic dual-role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis

Two systems are essential in humans for genome integrity, DNA repair and apoptosis. Cells that are defective in DNA repair tend to accumulate excess DNA damage. Cells defective in apoptosis tend to survive with excess DNA damage and thus allow DNA replication past DNA damages, causing mutations leading to carcinogenesis. It has recently become apparent that key proteins which contribute to cellular survival by acting in DNA repair become executioners in the face of excess DNA damage.Five major DNA repair pathways are homologous recombinational repair (HRR), non-homologous end joining (NHEJ), nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). In each of these DNA repair pathways, key proteins occur with dual functions in DNA damage sensing/repair and apoptosis. Proteins with these dual roles occur in: (1) HRR (BRCA1, ATM, ATR, WRN, BLM, Tip60 and p53); (2) NHEJ (the catalytic subunit of DNA-PK); (3) NER (XPB, XPD, p53 and p33(ING1b)); (4) BER (Ref-1/Ape, poly(ADP-ribose) polymerase-1 (PARP-1) and p53); (5) MMR (MSH2, MSH6, MLH1 and PMS2). For a number of these dual-role proteins, germ line mutations causing them to be defective also predispose individuals to cancer. Such proteins include BRCA1, ATM, WRN, BLM, p53, XPB, XPD, MSH2, MSH6, MLH1 and PMS2.     

Human DNA polymerase beta initiates DNA synthesis during long-patch repair of reduced AP sites in DNA

Simple base damages are repaired through a short-patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase beta (Pol beta) is responsible for the repair synthesis in this pathway and also removes a 5'-sugar phosphate residue by catalyzing a beta-elimination reaction. How ever, some DNA lesions that render deoxyribose resistant to beta-elimination are removed through a long-patch repair pathway that involves strand displacement synthesis and removal of the generated flap by specific endonuclease. Three human DNA polymerases (Pol beta, Pol delta and Pol epsilon) have been proposed to play a role in this pathway, however the identity of the polymerase involved and the polymerase selection mechanism are not clear. In repair reactions catalyzed by cell extracts we have used a substrate containing a reduced apurinic/apyrimidinic (AP) site resistant to beta-elimination and inhibitors that selectively affect different DNA polymerases. Using this approach we find that in human cell extracts Pol beta is the major DNA polymerase incorporating the first nucleotide during repair of reduced AP sites, thus initiating long-patch base excision repair synthesis.     

Recombinational repair of hydrogen peroxide-induced damages in DNA of phage T4

Recently, hydrogen peroxide and its free-radical product, the hydroxyl radical (OH.) have been identified as major sources of DNA damage in living organisms. They occur as ubiquitous metabolic by-products and, in humans, cause several thousand damages in a cell's DNA per day. They are thought to be a major source of DNA damage leading to aging and cancer in multicellular organisms. This raises two questions. First, what pathways are used in repair of DNA damages caused by H2O2 and OH.? Second, a new theory has been proposed that sexual reproduction (sex) evolved to promote repair of DNA in the germ line of organisms. If this theory is correct, then the type of repair specifically available during the sexual process should be able to deal with important natural lesions such as those produced by H2O2 and OH. . Does this occur? We examined repair of hydrogen peroxide damage to DNA, using a standard bacteriophage T4 test system in which sexual reproduction is either permitted or not permitted. Post-replication recombinational repair and denV-dependent excision repair are not dependent on sex. Both of these processes had little or no effect on lethal H2O2 damage. Also, an enzyme important in repair of H2O2-induced DNA damage in the E. coli host cells, exonuclease III, was not utilized in repair of lethal H2O2 damage to the phage. However, multiplicity reactivation, a recombinational form of repair depending on the sexual interaction of two or more of the bacteriophage, was found to repair lethal H2O2 damages efficiently. Our results lend support to the repair hypothesis of sex. Also the homology-dependent recombinational repair utilized in the phage sexual process may be analogous to the homology-dependent recombination which is widespread in diploid eucaryotes. The recombinational repair pathway found in phage T4 may thus be a widely applicable model for repair of the ubiquitous DNA damage caused by endogenous oxidative reactions.     

The role of irreparable DNA damage in initiating chromosomal mutagenesis in Chinese hamster cells irradiated with gamma and secondary proton radiation with an energy of 70 GeV

The study on the kinetics of DNA injury repair in Chinese hamster cells exposed to gamma and secondary proton radiation (70 GeV) has demonstrated the absence of distinctions in the kinetics of repair of rapidly repaired damages, a decreased rate of repair of slowly repaired lesions, and an increased residual irreparable damage induced by secondary radiation that reliably correlates with the RBE value estimated by the cytogenetic effect.     

Mystery of DNA repair: the role of the MRN complex and ATM kinase in DNA damage repair

Genomes are subject to a number of exogenous or endogenous DNA-damaging agents that cause DNA double-strand breaks (DSBs). These critical DNA lesions can result in cell death or a wide variety of genetic alterations, including deletions, translocations, loss of heterozygosity, chromosome loss, or chromosome fusions, which enhance genome instability and can trigger carcinogenesis. The cells have developed an efficient mechanism to cope with DNA damages by evolving the DNA repair machinery. There are 2 major DSB repair mechanisms: nonhomologous end joining (NHEJ) and homologous recombination (HR). One element of the repair machinery is the MRN complex, consisting of MRE11, RAD50 and NBN (previously described as NBS1), which is involved in DNA replication, DNA repair, and signaling to the cell cycle checkpoints. A number of kinases, like ATM (ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad-3-related), and DNA PKcs (DNA protein kinase catalytic subunit), phosphorylate various protein targets in order to repair the damage. If the damage cannot be repaired, they direct the cell to apoptosis. The MRN complex as well as repair kinases are also involved in telomere maintenance and genome stability. The dysfunction of particular elements involved in the repair mechanisms leads to genome instability disorders, like ataxia telangiectasia (A-T), A-T-like disorder (ATLD) and Nijmegen breakage syndrome (NBS). The mutated genes responsible for these disorders code for proteins that play key roles in the process of DNA repair. Here we present a detailed review of current knowledge on the MRN complex, kinases engaged in DNA repair, and genome instability disorders.     

The repair of bleomycin-induced DNA damage and its relationship to chromosome aberration repair.

Previous studies using the technique of premature chromosome condensation indicated that nearly one-half of the bleomycin-induced chromatid breaks and gaps in CHO cells could be repaired within 1 h (repair starting at 30 min) after treatment. Cycloheximide and streptovitacin A (but not hydroxyurea or hycanthone) inhibited chromosome repair. The purpose of this study was to measure the kinetics of DNA repair after bleomycin treatment using the alkaline elution technique and to determine whether various inhibitors could block this repair. After bleomycin treatment, the major proportion of the repair of DNA damage occurred within 15 min, with significant repair evident by 2 min. This fast repair component was inhibited by 0.2% EDTA. A slower repair component was observed to occur up to 60 min after bleomycin treatment. None of the inhibitors tested were found to have a significant effect on the repair of bleomycin damage at the DNA level. Since chromosome breaks were observed not to begin repair until after 30 min while over 50% of the DNA was repaired by 15 min, these results suggest that the DNA lesions that are repaired quickly are not important in the formation of chromosome aberrations. Further, since cycloheximide and streptovitacin A blocked chromosome repair but had little measurable effect on DNA repair, these results suggest that the DNA lesions responsible for chromosome damage represent only a small proportion of the total DNA lesions produced by bleomycin.     

Fragile DNA repair mechanism reduces ageing in multicellular model

DNA damages, as well as mutations, increase with age. It is believed that these result from increased genotoxic stress and decreased capacity for DNA repair. The two causes are not independent, DNA damage can, for example, through mutations, compromise the capacity for DNA repair, which in turn increases the amount of unrepaired DNA damage. Despite this vicious circle, we ask, can cells maintain a high DNA repair capacity for some time or is repair capacity bound to continuously decline with age? We here present a simple mathematical model for ageing in multicellular systems where cells subjected to DNA damage can undergo full repair, go apoptotic, or accumulate mutations thus reducing DNA repair capacity. Our model predicts that at the tissue level repair rate does not continuously decline with age, but instead has a characteristic extended period of high and non-declining DNA repair capacity, followed by a rapid decline. Furthermore, the time of high functionality increases, and consequently slows down the ageing process, if the DNA repair mechanism itself is vulnerable to DNA damages. Although counterintuitive at first glance, a fragile repair mechanism allows for a faster removal of compromised cells, thus freeing the space for healthy peers. This finding might be a first step toward understanding why a mutation in single DNA repair protein (e.g. Wrn or Blm) is not buffered by other repair proteins and therefore, leads to severe ageing disorders.     

The role of nucleotide excision repair of Escherichia coli in repair of spontaneous and gamma-radiation-induced DNA damage in the lacZalpha gene

Base excision repair (BER) is a very important repair mechanism to remove oxidative DNA damage. A major oxidative DNA damage after exposure to ionizing radiation is 7,8-dihydro-8-oxoguanine (8oxoG). 8oxoG is a strong mutagenic lesion, which may cause G:C to T:A transversions if not repaired correctly. Formamidopyrimidine-DNA glycosylase (Fpg), a repair enzyme which is part of BER, is the most important enzyme to repair 8oxoG. In the past years, evidence evolved that nucleotide excision repair (NER), a repair system originally thought to repair only bulky DNA lesions, can also repair some oxidative DNA damages. Examples of DNA damages which are recognized by NER are thymine glycol and abasic sites (AP sites). The main objective of this study is to determine if NER can act as a backup system for the repair of spontaneous and gamma-radiation-induced damages when Fpg is deficient. For that purpose, the effect of a NER-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZ gene was determined, using double-stranded (ds) M13 DNA, with the lacZalpha gene inserted as mutational target sequence. Subsequently the DNA was transfected into a fpg(-)uvrA(-) Escherichia coli strain (BH420) and the mutational spectra were compared with the spectra of a fpg(-) E. coli strain (BH410) and a wild type E. coli strain (JM105), which were determined in an earlier study. Furthermore, to examine effects which are caused by UvrA-deficiency, and not by Fpg-deficiency, the spontaneous and gamma-radiation-induced mutation spectra of an E. coli strain in which only UvrA is deficient (BH430) were also determined and compared with a wild type E. coli strain (JM105). The results of this study indicate that if only UvrA is deficient, there is an increase in spontaneous G:C to T:A transversions as compared to JM105 and a decrease in A:T to G:C transitions. The gamma-radiation-induced mutation spectrum of BH420 (fpg(-)uvrA(-)) shows a significant decrease in G:C to A:T and G:C to T:A mutations, as compared to BH410 where only Fpg is deficient. Based on these results, we conclude that in our experiments NER is not acting as a backup system if Fpg is deficient. Instead, NER seems to make mistakes, leading to the formation of mutations.     

The characteristics of the realization of cytogenetic damage in mammalian and plant cells exposed to low doses of radiation]

A complicated character of the cytogenetic injury dependence upon radiation dose was revealed after low-level gamma irradiation of Vicia faba seedlings and Chinese hamster fibroblasts. The dependence was linear with low-level secondary exposure to 70 GeV protons. The authors discuss a threshold nature of induction of the cytogenetic damage repair responsible for a high outcome of damages under the effect of low-level gamma radiation.     

Role of excision mechanisms of DNA repair in induction of apoptosis

DNA repair and apoptosis lead to principally different final results: the first mechanism removes damages from DNA, restoring genome integrity; the second mechanism eliminates potentially dangerous cells harboring DNA lesions. The cells deficient in mismatch repair (MMR) demonstrate inceased resistance (viability) to DNA-damaging agents due to decreased ability to undergo apoptosis. This means that mechanism of MMR both restores structure of DNA and generates a signal for apoptosis. DNA breaks and single strand gaps, which are temporarily produced by excison mechanism during DNA repair, are suggested to be the initial signals for apoptosis. However pathway involved in such signaling at least partially is independent of p53 function.     

Modulation of DNA damage/DNA repair capacity by XPC polymorphisms

XPC, a key protein in the nucleotide excision repair (NER) pathway, recognizes damaged DNA and initiates NER. Genetic variations in the XPC gene might be associated with altered DNA repair capacities (DRC). In this study, we genotyped three XPC polymorphisms, Ala499Val (C-->T), PAT (-/+) and Lys939Gln (A-->C), and measured the DNA damage/DRC by alkaline comet assay challenged by BPDE and gamma-radiation in 476 healthy subjects. We also evaluated the associations between DNA damage/DRC and genotypes of XPC polymorphisms. Compared with the XPC Lys939Gln homozygous wild type (AA) subjects, subjects with the variant alleles (AC and CC) had significantly higher DNA damages induced by BPDE (Median and 95% confidence interval [CI]: 3.16 (3.01-3.44) vs. 2.88 (2.51-3.05), P=0.01), and gamma-radiation (4.18 (3.94-4.44) vs. 3.71 (3.49-4.04), P=0.01). However, subjects with the variant alleles (CT and TT) of Ala499Val exhibited a 8.6% and 13.1% decrease in DNA damages induced by BPDE (P=0.05) and gamma-radiation (P=0.001), respectively. Significant correlations were found between genotypes and induced DNA damages in XPC Lys939Gln (For BPDE: R=0.12, P=0.01; for gamma-radiation: R=0.094, P=0.046) and Ala499Val (For BPDE: R=-0.11, P=0.03; for gamma-radiation: R=-0.16, P=0.0009). The haplotypes "T-A" (in the order of Ala499Val-PAT-Lys939Gln) was associated with the lowest DNA damages. Our results suggested that the DRC of host cells might be modulated by specific XPC polymorphisms.     

Damage recognition in nucleotide excision DNA repair

Nucleotide excision repair (NER) is a highly versatile DNA repair process. Its ability to repair a large number of different damages with the same subset of recognition factors requires structural tools for damage recognition that are both broad and very accurate. Over the past few years detailed structural information on damage recognition factors from eukaryotic and prokaryotic NER has emerged. These structures shed light on the toolkit utilized in the damage recognition process and help explain the broad substrate specificity of NER.     

Nickel chloride inhibits the DNA repair of UV-treated but not methyl methanesulfonate-treated chinese hamster ovary cells

Nickel, a human carcinogen, has been shown to enhance the cytotoxicity, mutagenicity, and sister-chromatid exchanges (SCE) induced by ultraviolet (UV) light but not by methyl methanesulfonate (MMS). To verify that the cocytotoxicity and cogenotoxicity of nickel are correlated with its inhibition on DNA repair, the effects of nickel on the DNA repair induced by UV and by MMS have been investigated. Our analyses of DNA repair of single-strand breaks by alkaline elution and alkaline sucrose sedimentation indicate that nickel inhibited the DNA repair in UV-treated, but not in MMS-treated cells. Therefore, the inhibition of DNA repair seems to play an important role in the cocytotoxicity and comutagenicity of nickel. However, the inhibition of DNA repair seems not to play a decisive role in enhancing SCE, because we have previously shown that arsenite inhibits the UV-induced DNA repair, but has no enhancing effect on the UV-induced SCE. Our results also show that nickel had obvious inhibitory effects on DNA ligation and postreplication repair, but had no apparent effect on nucleotide excision and DNA polymerization in the UV repair. The results of the DNA ligation inhibition by nickel in UV but not in MMS repair suggest that different ligases are used in the DNA repair of UV- and MMS-induced damages.     

Inhibition of repair of radiation-induced DNA damage by thermal shock in Chinese hamster ovary cells

The effect of exposure to elevated temperatures (41-45 degrees C) on the repair of radiation-induced DNA strand breaks was measured in monolayer cultured Chinese hamster ovary (CHO) cells. Prior exposure of cells to temperatures between 43 and 45 degrees C resulted in significant decreases in the rate of repair of DNA damage. Exposure to 45 degrees C for 15 min slowed the rate of DNA repair to 0.17 of the control repair rate. The To for inactivation of DNA repair was observed to be 34, 13 and 6 min at 43, 44 and 45 degrees C, respectively. Stepdown-heating (45 degrees C for 15 min followed by repair at 41 degrees C) resulted in greater inhibition of DNA repair (0.11 of the control rate) than was observed after acute heating alone. Repair at 41 degrees C was observed to proceed in unheated cells at a faster rate than at 37 degrees C. An Arrhenius analysis of the inactivation kinetics of DNA repair between 43 and 45 degrees C indicated an activation energy of 140 kcal mol-1 of protein for the inhibition of DNA repair. In general, the results were inconsistent with either a retardation of the DNA repair rate or an increase in unrepaired DNA lesions being responsible for heat-induced radiosensitization.     

A study of DNA repair in the thymocytes of irradiated rats using a spectrofluorometric method

In studying DNA repair in thymocytes of irradiated rats it was shown that the increase in radiation dose from 2 to 20 Gy made DNA damages increase in number and caused changes in their spectrum and growth of irreparable damages. The one-hour study of DNA repair process exhibited its fast, median and slow phases.