API

Event: 155

Key Event Title

?

N/A, Insufficient or incorrect DNA repair

Short name

?

N/A, Insufficient or incorrect DNA repair

Key Event Component

?

Process Object Action
DNA repair deoxyribonucleic acid functional change

Key Event Overview


AOPs Including This Key Event

?


Stressors

?



Level of Biological Organization

?

Biological Organization
Cellular

Cell term

?

Cell term
eukaryotic cell


Organ term

?



Taxonomic Applicability

?

Term Scientific Term Evidence Link
mouse Mus musculus Strong NCBI
rat Rattus norvegicus Moderate NCBI
Syrian golden hamster Mesocricetus auratus Moderate NCBI
Homo sapiens Homo sapiens Strong NCBI

Life Stages

?



Sex Applicability

?



How This Key Event Works

?


DNA lesions may result from the formation of DNA adducts (i.e., covalent modification of DNA by chemicals), or by the action of agents such as radiation that may produce strand breaks or modified nucleotides within the DNA molecule. These DNA lesions are repaired through several mechanistically distinct pathways that can be categorized as follows.

1) Damage reversal acts to reverse the damage without breaking any bonds within the sugar phosphate backbone of the DNA. The most prominent enzymes associated with damage reversal are photolyases (Sancar, 2003) that can repair UV dimers in some organisms, and O6-alkylguanine-DNA alkyltransferase (AGT) (Pegg 2011) and oxidative demethylases (Sundheim et al., 2008), which can repair some types of alkylated bases.

2) Excision repair involves the removal of a damaged nucleotide(s) through cleavage of the sugar phosphate backbone followed by re-synthesis of DNA within the resultant gap. Excision repair of DNA lesions can be mechanistically divided into base excision repair (BER) (Dianov and Hübscher, 2013), in which the damaged base is removed by a damage-specific glycosylase prior to incision of the phosphodiester backbone at the resulting abasic site, and nucleotide excision repair (NER) (Schärer, 2013), in which the DNA strand containing the damaged nucleotide is incised at sites several nucleotides 5’ and 3’ to the site of damage, and a polynucleotide containing the damaged nucleotide is removed prior to DNA resynthesis within the resultant gap. A third form of excision repair is mismatch repair (MMR), which does not act on DNA lesions but does recognize mispaired bases resulting from replication errors. In MMR the strand containing the misincorporated base is removed prior to DNA resynthesis.

3) Double strand break repair (DSBR) is necessary to preserve genomic integrity when breaks occur in both strands of a DNA molecule. There are two major pathways for DSBR: homologous recombination (HR), which operates primarily during S phase in dividing cells, and nonhomologous end joining (NHEJ), which can function in both dividing and non-dividing cells (Teruaki Iyama and David M. Wilson III, 2013).


Most DNA repair pathways are extremely efficient. However, in principal, all DNA repair pathways can be overwhelmed when the DNA lesion burden exceeds the capacity of a given DNA repair pathway to recognize and remove the lesion. Such DNA repair insufficiency may lead to toxicity or mutagenesis following DNA damage. Apart from extremely high DNA lesion burden, DNA insufficiency may arise through several different specific mechanisms. For example, during repair of DNA containing O6-alkylguanine adducts, AGT irreversibly binds a single O6-alkylguanine lesion and as a result is inactivated (this is termed suicide inactivation, as its own action causes it to become inactivated). Thus, the capacity of AGT to carry out alkylation repair can become rapidly saturated when the DNA repair rate exceeds the de novo synthesis of AGT (Pegg, 2011). A second mechanism relates to cell specific differences in the cellular levels or activity of some DNA repair proteins. For example, XPA is an essential component of the NER complex. The level of XPA that is active in NER is low in the testes, which may reduce the efficiency of NER in testes as compared to other tissues (Köberle et al., 1999). Likewise, both NER and BER have been reported to be deficient in cells lacking functional p53 (Adimoolam and Ford, 2003; Hanawalt et al., 2003; Seo and Jung, 2004). A third mechanism relates to the importance of the DNA sequence context of a lesion in its recognition by DNA repair enzymes. For example, 8-oxoguanine (8-oxoG) is repaired primarily by BER; the lesion is initially acted upon by a bifunctional glycosylase, OGG1, which carries out the initial damage recognition and excision steps of 8-oxoG repair. However, the rate of excision of 8-oxoG is modulated strongly by both chromatin components (Menoni et al., 2012) and DNA sequence context (Allgayer et al., 2013) leading to significant differences in the repair of lesions situated in different chromosomal locations.

DNA repair is also remarkably error-free. However, misrepair can arise during repair under some circumstances. DSBR is notably error prone, particularly when breaks are processed through NHEJ, during which partial loss of genome information is common at the site of the double strand break (Iyama and Wilson, 2013). Excision repair pathways require the resynthesis of DNA and rare DNA polymerase errors during gap resynthesis will result in mutations (Brown et al., 2011). Errors may also arise during gap resynthesis when the strand that is being used as a template for DNA synthesis contains DNA lesions (Kozmin and Jinks-Robertson, 2013). In addition, it has been shown that sequences that contain tandemly repeated sequences, such as CAG triplet repeats, are subject to expansion during gap resynthesis that occurs during BER of 8-oxoG damage (Liu et al., 2009).


How It Is Measured or Detected

?


There is no test guideline for this event. The event is usually inferred from measuring the retention of DNA adducts or the creation of mutations as a measure of lack of repair or incorrect repair. These ‘indirect’ measures of its occurrence are crucial to determining the mechanisms of genotoxic chemicals and for regulatory applications (i.e., determining the best approach for deriving a point of departure). More recently, a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) has been developed to directly measures the ability of human cells to repair plasmid reporters (Nagel et al., 2014).

INDIRECT MEASUREMENT

In somatic and spermatogenic cells, measurement of DNA repair is usually inferred by measuring DNA adduct formation/removal. Insufficient repair is inferred from the retention of adducts and from increasing adduct formation with dose. Insufficient DNA repair is also measured by the formation of increased numbers of mutations and alterations in mutation spectrum. The methods will be specific to the type of DNA adduct that is under study.

Some EXAMPLES are given below for alkylated DNA.

DOSE-RESPONSE CURVE FOR ALKYL ADDUCTS/MUTATIONS: It is important to consider that some adducts are not mutagenic at all because they are very effectively repaired. Others are effectively repaired, but if these repair processes become overwhelmed mutations begin to occur. The relationship between exposure to mutagenic agents and the presence of adducts (determined as adducts per nucleotide) provide an indication of whether the removal of adducts occurs, and whether it is more efficient at low doses. A sub-linear DNA adduct curve suggests that less effective repair occurs at higher doses (i.e., repair processes are becoming saturated). A sub-linear shape for the dose-response curves for mutation induction is also suggestive of repair of adducts at low doses, followed by saturation of repair at higher doses. Measurement of a clear point of inflection in the dose-response curve for mutations suggests that repair does occur, at least to some extent, but reduced repair efficiency arises above the breakpoint. A lack of increase in mutation frequencies (i.e., flat line for dose-response) for a compound showing a dose-dependent increase in adducts would imply that the adducts formed are either not mutagenic or are effectively repaired.

RETENTION OF ALKYL ADDUCTS: Alkylated DNA can be found in cells long after exposure has occurred. This indicates that repair has not effectively removed the adducts. For example, DNA adducts have been measured in hamster and rat spermatogonia several days following exposure to alkylating agents, indicating lack of repair (Seiler et al., 1997; Scherer et al., 1987).

MUTATION SPECTRUM: Shifts in mutation spectrum (i.e., the specific changes in the DNA sequence) following a chemical exposure (relative to non-exposed mutation spectrum) indicates that repair was not operating effectively to remove specific types of lesions. The shift in mutation spectrum is indicative of the types of DNA lesions (target nucleotides and DNA sequence context) that were not repaired. For example, if a greater proportion of mutations occur at guanine nucleotides in exposed cells, it can be assumed that the chemical causes DNA adducts on guanine that are not effectively repaired.


DIRECT MEASUREMENT

Nagel et al. (2014) we developed a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) to measures the ability of human cells to repair plasmid reporters. These reporters contain different types and amounts of DNA damage and can be used to measure repair through by NER, MMR, BER, NHEJ, HR and MGMT.


Evidence Supporting Taxonomic Applicability

?


The retention of adducts has been directly measured in many different types of eukaryotic somatic cells (in vitro and in vivo). In male germ cells, work has been done on hamsters, rats and mice. The accumulation of mutation and changes in mutation spectrum has been measured in mice and human cells in culture. Theoretically, saturation of DNA repair occurs in every species (prokaryotic and eukaryotic). The principles of this work were established in prokaryotic models. Nagel et al. (2014) have produced an assay that directly measures DNA repair in human cells in culture.


References

?


Adimoolam, S. and J.M. Ford (2003), "p53 and regulation of DNA damage recognition during nucleotide excision repair" DNA Repair (Amst), 2(9): 947-54.

Allgayer, J., N. Kitsera, C. von der Lippen, B. Epe and A. Khobta (2013), "Modulation of base excision repair of 8-oxoguanine by the nucleotide sequence", Nucleic Acids Res., 41(18): 8559-8571.

Beranek, D.T. (1990), "Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents", Mutation Research, 231(1): 11-30.

Bronstein, S.M., J.E. Cochrane, T.R. Craft, J.A. Swenberg and T.R. Skopek (1991), "Toxicity, mutagenicity, and mutational spectra of N-ethyl-N-nitrosourea in human cell lines with different DNA repair phenotypes", Cancer Research, 51(!9): 5188-5197.

Bronstein, S.M., T.R. Skopek and J.A. Swenberg (1992), "Efficient repair of O6-ethylguanine, but not O4-ethylthymine or O2-ethylthymine, is dependent upon O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair activities in human cells", Cancer Research, 52(7): 2008-2011.

Brown, J.A., L.R. Pack, L.E. Sanman and Z. Suo (2011), "Efficiency and fidelity of human DNA polymerases λ and β during gap-filling DNA synthesis", DNA Repair (Amst)., 10(1):24-33.

Dianov, G.L. and U. Hübscher (2013), "Mammalian base excision repair: the forgotten archangel", Nucleic Acids Res., 41(6):3483-90.

Douglas, G.R., J. Jiao, J.D. Gingerich, J.A. Gossen and L.M. Soper (1995), "Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice", Proceedings of the National Academy of Sciences of the United States of America, 92(16):7485-7489.

Hanawalt, P.C., J.M. Ford and D.R. Lloyd (2003), "Functional characterization of global genomic DNA repair and its implications for cancer", Mutation Research, 544(2-3): 107–114.

Iyama, T. and D.M. Wilson III (2013), "DNA repair mechanisms in dividing and non-dividing cells", DNA Repair, 12(8): 620– 636.

Köberle, B., J.R. Masters, J.A. Hartley and R.D. Wood (1999), "Defective repair of cisplatin-induced DNA damage caused by reduced XPA protein in testicular germ cell tumours", Curr. Biol., 9(5):273-6.

Kozmin, S.G. and S. Jinks-Robertson S. 2013. The mechanism of nucleotide excision repair-mediated UV-induced mutagenesis in nonproliferating cells. Genetics. 193(3): 803-17.

Liu, Y., R. Prasad, W.A. Beard, E.W. Hou, J.K. Horton, C.T. McMurray and S.H. Wilson (2009), "Coordination between polymerase beta and FEN1 can modulate CAG repeat expansion", J. Biol. Chem., 284(41): 28352-28366.

Menoni, H., M.S. Shukla, V. Gerson, S. Dimitrov and D. Angelov (2012), "Base excision repair of 8-oxoG in dinucleosomes", Nucleic Acids Res. , 40(2): 692-700.

Nagel, Z.D., C.M. Margulies, I.A. Chaim, S.K. McRee, P. Mazzucato, A.A. Ahmad, R.P. Abo, V.L. Butty, A.L. Forget and L.D. Samson (2014), "Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis", Proc. Natl. Acad. Sci. USA. 111(18):E1823-32.

O’Brien, J.M., M. Walker, A. Sivathayalan, G.R. Douglas, C.L. Yauk, and F. Marchetti (2015), "Sublinear response in lacZ mutant frequency of Muta™ Mouse spermatogonial stem cells after low dose subchronic exposure to N-ethyl-N-nitrosourea", Environ. Mol. Mutagen., 56(4): 347-55.

Pegg, A.E. (2011), "Multifaceted roles of alkyltransferase and related proteins in DNA repair, DNA damage, resistance to chemotherapy, and research tools", Chem. Res. Toxicol., 4(5): 618-39.

Sancar, A. (2003), "Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors", Chem Rev., 103(6): 2203-37.

Schärer, O.D. (2013), "Nucleotide excision repair in eukaryotes", Cold Spring Harb. Perspect. Biol., 5(10): a012609.

Scherer, E., A.A. Jenner and L. den Engelse (1987), "Immunocytochemical studies on the formation and repair of O6-alkylguanine in rat tissues", IARC Sci Publ., 84: 55-8.

Seiler, F., K. Kamino, M. Emura, U. Mohr and J. Thomale (1997), "Formation and persistence of the miscoding DNA alkylation product O6-ethylguanine in male germ cells of the hamster", Mutat Res., 385(3): 205-211.

Shelby, M.D. and K.R. Tindall (1997), "Mammalian germ cell mutagenicity of ENU, IPMS and MMS, chemicals selected for a transgenic mouse collaborative study", Mutation Research, 388(2-3): 99-109.

Seo, Y.R. and H.J. Jung (2004), "The potential roles of p53 tumor suppressor in nucleotide excision repair (NER) and base excision repair (BER)", Exp. Mol. Med., 36(6): 505-509.

Sundheim, O., V.A. Talstad, C.B. Vågbø, G. Slupphaug and H.E. Krokan (2008), "AlkB demethylases flip out in different ways", DNA Repair (Amst)., 7(11): 1916-1923.

van Zeeland, A.A., A. de Groot and A. Neuhäuser-Klaus (1990), "DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis", Mutat. Res., 231(1): 55-62.