Mechanisms of human cell protection associated with genetic polymorphism

Systems ensuring protection of human cells against endogenous and exogenous mutagenic factors are considered in terms of genetic polymorphism. Some protection mechanisms are described, including those connected with capturing free radicals, biotransformation of xenobiotics, excision repair of DNA damage (excision of nitrous bases, nucleotides, mismatch repair). A special section is devoted to some issues of using antimutagens in context of genetic polymorphism. The problem of adaptive response is discussed, providing evidence for independence (in some cases) of DNA repair systems and the formation of adaptive response. Some results of the author obtained many years ago but still relevant are presented.     

How does a cell repair damaged DNA?

DNA in living cells is constantly subjected to different chemical and physical factors of the environment and to cell metabolites. Some changes altering DNA structure occur spontaneously. This raises the potential danger of harmful mutations that could be transmitted to offspring. To avoid the danger of mutations and changing genetic information, a cell is capable to switch on multiple mechanisms of DNA repair that remove damage and restore native structure. In many cases, removal of the same damage may involve several alternative pathways; this is very important for DNA repair under the most unfavorable conditions. This review summarizes data about all known mechanisms of eukaryotic DNA repair including excision repair (base excision repair and nucleotide excision repair), mismatch repair, repair of double-strand breaks, and cross-link repair. Special attention is given to the regulation of excision repair by different proteins—proliferating cell nuclear antigen (PCNA), p53, and proteasome. The review also highlights problem of bypassing irremovable lesions in DNA.Translated from Biokhimiya, Vol. 70, No. 3, 2005, pp. 341–359.Original Russian Text Copyright © 2005 by Sharova.     

Base Excision Repair of DNA: Glycosylases

The review considers the role of base excision repair in maintaining the constancy of genetic information in the cell. The genetic control and biochemical mechanism are described for the first stage of base excision repair, which is catalyzed by specific enzymes, DNA glycosylases.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 725–735.Original Russian Text Copyright © 2005 by Korolev.     

Adaptive response to gamma radiation in mammalian cells proficient and deficient in components of nucleotide excision repair

Cells preconditioned with low doses of low-linear energy transfer (LET) ionizing radiation become more resistant to later challenges of radiation. The mechanism(s) by which cells adaptively respond to radiation remains unclear, although it has been suggested that DNA repair induced by low doses of radiation increases cellular radioresistance. Recent gene expression profiles have consistently indicated that proteins involved in the nucleotide excision repair pathway are up-regulated after exposure to ionizing radiation. Here we test the role of the nucleotide excision repair pathway for adaptive response to gamma radiation in vitro. Wild-type CHO cells exhibited both greater survival and fewer HPRT mutations when preconditioned with a low dose of gamma rays before exposure to a later challenging dose. Cells mutated for ERCC1, ERCC3, ERCC4 or ERCC5 did not express either adaptive response to radiation; cells mutated for ERCC2 expressed a survival adaptive response but no mutation adaptive response. These results suggest that some components of the nucleotide excision repair pathway are required for phenotypic low-dose induction of resistance to gamma radiation in mammalian cells.     

Lymphocyte DNA adducts and polymorphism in the DNA repair enzyme XPD

The effect of genetic polymorphism of DNA repair enzyme on the DNA adduct levels was evaluated in this study. We explored the relationship between polymorphism in the nucleotide excision repair enzyme XPD and DNA adduct levels in lymphocytes. Lymphocyte DNA adducts were measured by a ³²     

Lymphocyte DNA adducts and polymorphism in the DNA repair enzyme XPD

The effect of genetic polymorphism of DNA repair enzyme on the DNA adduct levels was evaluated in this study. We explored the relationship between polymorphism in the nucleotide excision repair enzyme XPD and DNA adduct levels in lymphocytes. Lymphocyte DNA adducts were measured by a ³²     

Alternative pathways for the in vivo repair of O6-alkylguanine and O4-alkylthymine in Escherichia coli: the adaptive response and nucleotide excision repair.

The in vivo removal of three different O-alkylated bases from DNA was measured in Escherichia coli. Using monoclonal antibodies specific for O6-methylguanine, O6-ethylguanine and O4-ethylthymine we have monitored the removal of these lesions from six different strains to assess the relative contributions of the adaptive response and of nucleotide excision repair. During the first hour after DNA alkylation, O6-methylguanine, O6-ethylguanine and O4-ethylthymine lesions were repaired almost exclusively by nucleotide excision, except when the adaptive response was being constitutively expressed. In wild-type E. coli the adaptive response began to contribute to O6-methylguanine repair about one hour after alkylation, the time required for the full induction of the ada DNA methyltransferase. In contrast, the adaptive response did not play such a large role in the repair of O6-ethylguanine and O4-ethylthymine in wild-type E. coli, presumably because DNA ethylation damage is a poor inducer of the adaptive response; possible reasons for this poor induction are discussed. The repair of all three O-alkylated lesions was virtually absent in ada- uvr- bacteria suggesting that no alternative pathway is available for their repair, at least during the first two hours after alkylation. When the repair of O-alkylated bases was compromised by an ada- or by a uvr- mutation, the bacteria became more sensitive to alkylation induced killing and mutation.     

Cellular heterogeneity in DNA alkylation repair increases population genetic plasticity

DNA repair mechanisms fulfil a dual role, as they are essential for cell survival and genome maintenance. Here, we studied how cells regulate the interplay between DNA repair and mutation. We focused on the adaptive response that increases the resistance of Escherichia coli cells to DNA alkylation damage. Combination of single-molecule imaging and microfluidic-based single-cell microscopy showed that noise in the gene activation timing of the master regulator Ada is accurately propagated to generate a distinct subpopulation of cells in which all proteins of the adaptive response are essentially absent. Whereas genetic deletion of these proteins causes extreme sensitivity to alkylation stress, a temporary lack of expression is tolerated and increases genetic plasticity of the whole population. We demonstrated this by monitoring the dynamics of nascent DNA mismatches during alkylation stress as well as the frequency of fixed mutations that are generated by the distinct subpopulations of the adaptive response. We propose that stochastic modulation of DNA repair capacity by the adaptive response creates a viable hypermutable subpopulation of cells that acts as a source of genetic diversity in a clonal population.     

Analysis of the polymorphisms XRCC1Arg194Trp and XRCC1Arg399Gln in gliomas

XRCC genes (X-ray cross-complementing group) were discovered mainly for their roles in protecting mammalian cells against damage caused by ionizing radiation. Studies determined that these genes are important in the genetic stability of DNA. Although the loss of some of these genes does not necessarily confer high levels of sensitivity to radiation, they have been found to represent important components of various pathways of DNA repair. To ensure the integrity of the genome, a complex system of DNA repair was developed. Base excision repair is the first defense mechanism of cells against DNA damage and a major event in preventing mutagenesis. Repair genes may play an important role in maintaining genomic stability through different pathways that are mediated by base excision. In the present study, we examined XRCC1Arg194Trp and XRCC1Arg399Gln polymorphism using PCR-RFLP in 80 astrocytoma and glioblastoma samples. Patients who had the allele Trp of the XRCC1Arg194Trp polymorphism had an increased risk of tumor development (OR = 8.80; confidence interval at 95% (95%CI) = 4.37-17.70; P < 0.001), as did the allele Gln of XRCC1Arg399Gln (OR = 1.01; 95%CI = 0.53-1.93; P = 0.971). Comparison of overall survival of patients did not show significant differences. We suggest that XRCC1Arg194Trp and XRCC1Arg399Gln polymorphisms are involved in susceptibility for developing astrocytomas and glioblastomas.     

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.     

The interplay of DNA polymerase λ in diverse DNA damage repair pathways in higher plant genome in response to environmental and genotoxic stress factors

DNA repair mechanisms are essential for the maintenance of genomic stability, proper cellular function and survival for all organisms. Plants, with their intrinsic immobility, are vastly exposed to a wide range of environmental agents and also endogenous processes which frequently cause damage to DNA and impose genotoxic stress. Therefore, in order to survive under frequent and extreme environmental stress conditions, plants have developed a vast array of efficient and powerful DNA damage repair mechanisms to ensure rapid and precise repair of genetic material for maintaining genome stability and faithful transfer of genetic information over generations.¹ Recently, we have defined the role of DNA polymerase λ in repair of UV-B-induced photoproducts in Arabidopsis thaliana via nucleotide excision repair pathway.² Here, we have further discussed potential function of DNA polymerase λ in various DNA repair pathways in higher plant genome in response to environmental and genotoxic stress factors.     

Role of base excision repair DNA glycosylases in hereditary and infectious human diseases

DNA glycosylases are enzymes that initiate base excision repair, which removes damaged bases from cell DNA. Recent data demonstrate that some genetic variants of two human DNA glycosylases, MUTYH and OGG1, are associated with an increased risk of cancer. In addition, various DNA glycosylases are involved in protection from some neurodegenerative diseases, immune disorders, and virus infections. On the other hand, DNA glycosylases of pathogenic microorganisms help them to evade the host defense mechanisms. Thus, DNA glycosylases are considered to be both potential therapeutic agents and drug targets.     

Topical thymidine dinucleotide application protects against UVB-induced skin cancer in mice with DNA repair gene (Ercc1)-deficient skin

Topical application of thymidine dinucleotides (pTpT) provides some protection against the effects of UV on the skin, however, many details of the protective mechanism have yet to be elucidated. We have used mice with an epidermis-specific knockout for the nucleotide excision repair gene, Ercc1, to investigate the mechanisms of protection. pTpT offered no protection against the pronounced UV-induced short-term erythema and skin thickening responses that are characteristic of DNA repair-deficient skin. It also had no effect on UV-induced apoptosis in Ercc1-deficient cultured keratinocytes. However, in these short-term experiments in both skin and keratinocyte culture pTpT did cause a slight reduction in proliferation. pTpT application during a chronic UV irradiation protocol provided some protection from UVB-induced skin carcinogenesis in epidermis-specific Ercc1 knockout mice. The median tumour free survival time was increased in the pTpT-treated group and treated animals had fewer tumours. In addition, pTpT-treated animals developed fewer large inwardly growing skin lesions than untreated animals. Furthermore, the proliferation response was reduced in chronically irradiated, non-lesional pTpT-treated skin. We conclude that cancer protection by pTpT in our mice is not modulated by an upregulation of DNA repair, as protection appears to be independent of a functional nucleotide excision repair pathway. We hypothesise instead that protection by pTpT is due to a reduction in epidermal proliferation.     

Expression of Escherichia coli dam gene in Bacillus subtilis provokes DNA damage response: N6-methyladenine is removed by two repair pathways.

The dam gene of Escherichia coli encodes a DNA methyltransferase that methylates the N6 position of adenine in the sequence GATC. It was stably expressed from a shuttle vector in a repair- and recombination-proficient strain of Bacillus subtilis. In this strain the majority of plasmid DNA molecules was modified at dam sites whereas most chromosomal DNA remained unmethylated during exponential growth. During stationary phase the amount of unmethylated DNA increased, suggesting that methylated bases were being removed. An ultraviolet damage repair-deficient mutant (uvrB) contained highly methylated chromosomal and plasmid DNA. High levels of Dam methylation were detrimental to growth and viability of this mutant strain and some features of the SOS response were also induced. A mutant defective in the synthesis of adaptive DNA alkyltransferases and induction of the adaptive response (ada) also showed high methylation and properties similar to that of the dam gene expressing uvrB strain. When protein extracts from B. subtilis expressing the Dam methyltransferase or treated with N-methyl-N'-nitro-N-nitroso-guanidine were incubated with [3H]-labelled Dam methylated DNA, the methyl label was bound to two proteins of 14 and 9 kD. Some free N6-methyladenine was also detected in the supernatant of the incubation mixture. We propose that N6-methyladenine residues are excised by proteins involved in both excision (uvrB) and the adaptive response (ada) DNA repair pathways in B. subtilis.     

Congenital DNA repair deficiency results in protection against renal ischemia reperfusion injury in mice

Cockayne syndrome and other segmental progerias with inborn defects in DNA repair mechanisms are thought to be due in part to hypersensitivity to endogenous oxidative DNA damage. The accelerated aging-like symptoms of this disorder include dysmyelination within the central nervous system, progressive sensineuronal hearing loss and retinal degeneration. We tested the effects of congenital nucleotide excision DNA repair deficiency on acute oxidative stress sensitivity in vivo . Surprisingly, we found mouse models of Cockayne syndrome less susceptible than wild type animals to surgically induced renal ischemia reperfusion injury, a multifactorial injury mediated in part by oxidative damage. Renal failure-related mortality was significantly reduced in Csb^−/– mice, kidney function was improved and proliferation was significantly higher in the regenerative phase following ischemic injury. Protection from ischemic damage correlated with improved baseline glucose tolerance and insulin sensitivity and a reduced inflammatory response following injury. Protection was further associated with genetic ablation of a different Cockayne syndrome-associated gene, Csa . Our data provide the first functional in vivo evidence that congenital DNA repair deficiency can induce protection from acute stress in at least one organ. This suggests that while specific types of unrepaired endogenous DNA damage may lead to detrimental effects in certain tissues, they may at the same time elicit beneficial adaptive changes in others and thus contribute to the tissue specificity of disease symptoms.     

Impact of DNA repair genes polymorphism (XPD and XRCC1) on the risk of breast cancer in Egyptian female patients

The genes involved in DNA repair system play a crucial role in the protection against mutations. It has been hypothesized that functional deficiencies in highly conserved DNA repair processes resulting from polymorphic variation may increase genetic susceptibility to breast cancer (BC). The aim of the present study was to evaluate the association of genetic polymorphisms in 2 DNA repair genes, XPD (Asp312Asn) and XRCC1 (A399G), with BC susceptibility. We further investigated the potential combined effect of these DNA repair variants on BC risk. Both XPD (xeroderma pigmentosum group D) and XRCC1 (X-ray repair cross-complementing group 1) polymorphisms were characterized in 100 BC Egyptian females and 100 healthy women who had no history of any malignancy by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method and PCR with confronting two-pair primers (PCR-CTPP), using DNA from peripheral blood in a case control study. Our results revealed that the frequencies of AA genotype of XPD codon 312 polymorphism were significantly higher in the BC patients than in the normal individuals (P ≤ 0.003), and did not observe any association between the XRCC1 Arg399Gln polymorphism and risk of developing BC. Also, no association between both XPD Asp312Asn and XRCC1 A399G polymorphisms and the clinical characteristics of disease. Finally, the combination of AA(XPD) + AG(XRCC1) were significantly associated with BC risk. Our results suggested that, XPD gene is an important candidate gene for susceptibility to BC. Also, gene–gene interaction between XPD(AA) + XRCC1(AG) polymorphism may be associated with increased risk of BC in Egyptian women.     

Alternative Excision Repair Pathways

Alternative excision repair (AER) is a category of excision repair initiated by a single nick, made by an endonuclease, near the site of DNA damage, and followed by excision of the damaged DNA, repair synthesis, and ligation. The ultraviolet (UV) damage endonuclease in fungi and bacteria introduces a nick immediately 5′ to various types of UV damage and initiates its excision repair that is independent of nucleotide excision repair (NER). Endo IV-type apurinic/apyrimidinic (AP) endonucleases from Escherichia coli and yeast and human Exo III-type AP endonuclease APEX1 introduce a nick directly and immediately 5′ to various types of oxidative base damage besides the AP site, initiating excision repair. Another endonuclease, endonuclease V from bacteria to humans, binds deaminated bases and cleaves the phosphodiester bond located 1 nucleotide 3′ of the base, leading to excision repair. A single-strand break in DNA is one of the most frequent types of DNA damage within cells and is repaired efficiently. AER makes use of such repair capability of single-strand breaks, removes DNA damage, and has an important role in complementing BER and NER.NER and base excision repair (BER) are the major excision repair pathways present in almost all organisms. In NER, dual incisions are introduced, the damaged DNA between the incised sites is then removed, and DNA synthesis fills the single-stranded gap, followed by ligation. In BER, an AP site, formed by depurination or created by a base damage-specific DNA glycosylase, is recognized by an AP endonuclease that introduces a nick immediately 5′ to the AP site, followed by repair synthesis, removal of the AP site, and final ligation. Besides these two fundamental excision repair systems, investigators have found another category of excision repair—AER—an example of which is the excision repair of UV damage, initiated by an endonuclease called UV damage endonuclease (UVDE). UVDE introduces a single nick immediately 5′ to various types of UV lesions as well as other types of base damage, and this nick leads to the removal of the lesions by an AER process designated as UVDE-mediated excision repair (UVER or UVDR). Genetic analysis in Schizosaccharomyces pombe indicates that UVER provides cells with an extremely rapid removal of UV lesions, which is important for cells exposed to UV in their growing phase.Endo IV–type AP endonucleases from Escherichia coli and budding yeast and the Exo III–type human AP endonuclease APEX1 are able to introduce a nick at various types of oxidative base damage and initiate a form of excision repair that has been designated as nucleotide incision repair (NIR). Endonuclease V (ENDOV) from bacteria to humans recognizes deaminated bases, introduces a nick 1 nucleotide 3′ of the base, and leads to excision repair initiated by the nick. These endonucleases introduce a single nick near the DNA-damage site, leaving 3′-OH termini, and initiate repair of both the DNA damage and the nick. The mechanisms of AER may be similar to those of single-strand break (SSB) repair or BER except for the initial nicking process. However, how DNA damage is recognized determines the repair process within the cell. This article discusses the mechanisms and functional roles of AER. We begin with AER of UV damage, because genetic analysis has shown functional differences between this AER and NER in S. pombe.