Although DNA is the carrier of genetic information, it has limited chemical stability. Hydrolysis, oxidation and nonenzymatic methylation of DNA occur at significant rates in vivo, and are counteracted by specific DNA repair processes. The spontaneous decay of DNA is likely to be a major factor in mutagenesis, carcinogenesis and ageing, and also sets limits for the recovery of DNA fragments from fossils.

Instability and decay of the primary structure of DNA

Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction

Oxidative DNA damage is an inevitable consequence of cellular metabolism, with a propensity for increased levels following toxic insult. Although more than 20 base lesions have been identified, only a fraction of these have received appreciable study, most notably 8-oxo-2'deoxyguanosine. This lesion has been the focus of intense research interest and been ascribed much importance, largely to the detriment of other lesions. The present work reviews the basis for the biological significance of oxidative DNA damage, drawing attention to the multiplicity of proteins with repair activities along with a number of poorly considered effects of damage. Given the plethora of (often contradictory) reports describing pathological conditions in which levels of oxidative DNA damage have been measured, this review critically addresses the extent to which the in vitro significance of such damage has relevance for the pathogenesis of disease. It is suggested that some shortcomings associated with biomarkers, along with gaps in our knowledge, may be responsible for the failure to produce consistent and definitive results when applied to understanding the role of DNA damage in disease, highlighting the need for further studies.

Oxidative DNA damage: mechanisms, mutation, and disease

Faithful maintenance of the genome is crucial to the individual and to species. DNA damage arises from both endogenous sources such as water and oxygen and exogenous sources such as sunlight and tobacco smoke. In human cells, base alterations are generally removed by excision repair pathways that counteract the mutagenic effects of DNA lesions. This serves to maintain the integrity of the genetic information, although not all of the pathways are absolutely error-free. In some cases, DNA damage is not repaired but is instead bypassed by specialized DNA polymerases.

https://science.sciencemag.org/content/sci/286/5446/1897.full.pdf

Quality control by DNA repair.

The generation of reactive oxygen species may be both beneficial to cells, performing a function in inter- and intracellular signalling, and detrimental, modifying cellular biomolecules, accumulation of which has been associated with numerous diseases. Of the molecules subject to oxidative modification, DNA has received the greatest attention, with biomarkers of exposure and effect closest to validation. Despite nearly a quarter of a century of study, and a large number of base- and sugar-derived DNA lesions having been identified, the majority of studies have focussed upon the guanine modification, 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-OH-dG). For the most part, the biological significance of other lesions has not, as yet, been investigated. In contrast, the description and characterisation of enzyme systems responsible for repairing oxidative DNA base damage is growing rapidly, being the subject of intense study. However, there remain notable gaps in our knowledge of which repair proteins remove which lesions, plus, as more lesions identified, new processes/substrates need to be determined. There are many reports describing elevated levels of oxidatively modified DNA lesions, in various biological matrices, in a plethora of diseases; however, for the majority of these the association could merely be coincidental, and more detailed studies are required. Nevertheless, even based simply upon reports of studies investigating the potential role of 8-OH-dG in disease, the weight of evidence strongly suggests a link between such damage and the pathogenesis of disease. However, exact roles remain to be elucidated.

Oxidative DNA damage and disease: induction, repair and significance

8-oxoguanine (8-oxoG), ring-opened purines (formamidopyrimidines or Fapys), and other oxidized DNA base lesions generated by reactive oxygen species are often mutagenic and toxic, and have been implicated in the etiology of many diseases, including cancer, and in aging. Repair of these lesions in all organisms occurs primarily via the DNA base excision repair pathway, initiated with their excision by DNA glycosylase/AP lyases, which are of two classes. One class utilizes an internal Lys residue as the active site nucleophile, and includes Escherichia coli Nth and both known mammalian DNA glycosylase/AP lyases, namely, OGG1 and NTH1. E. coli MutM and its paralog Nei, which comprise the second class, use N-terminal Pro as the active site. Here, we report the presence of two human orthologs of E. coli mutM nei genes in the human genome database, and characterize one of their products. Based on the substrate preference, we have named it NEH1 (Nei homolog). The 44-kDa, wild-type recombinant NEH1, purified to homogeneity from E. coli, excises Fapys from damaged DNA, and oxidized pyrimidines and 8-oxoG from oligodeoxynucleotides. Inactivation of the enzyme because of either deletion of N-terminal Pro or Histag fusion at the N terminus supports the role of N-terminal Pro as its active site. The tissue-specific levels of NEH1 and OGG1 mRNAs are distinct, and S phase-specific increase in NEH1 at both RNA and protein levels suggests that NEH1 is involved in replication-associated repair of oxidized bases.

https://www.pnas.org/content/pnas/99/6/3523.full.pdf

Identification and characterization of a human DNA glycosylase for repair of modified bases in oxidatively damaged DNA.

Identification and characterization of a novel human DNA glycosylase for repair of cytosine-derived lesions.

One of the most prevalent lesions in DNA is the apurinic/apyrimidinic (AP) site, which is derived from the cleavage of the N-glycosyl bond by DNA glycosylase or by spontaneous depurination. AP sites are repaired by AP endonucleases during the process of base excision repair; however, an imbalance in this DNA repair system may cause mutations as well as cell death. We have established a sensitive and convenient slot-blot method to detect AP sites in genomic DNA using a novel aldehyde reactive probe (ARP), which reacts with the aldehydic group of ring-opened AP sites. The reaction of 1 mM of ARP with 15 microg of genomic DNA containing AP sites at 37 degrees C was completed within 1 min. The AP site-ARP complex was remarkably stable during incubation in TE buffer, even at 100 degrees C for 60 min. The sensitivity of this assay enables detection of 2.4 AP sites per 10(7) bases. By using this ARP-slot-blot assay, the rate of spontaneous depurination of calf thymus DNA was determined. Under physiological conditions, AP sites were increased at 1.54 AP sites/10(6) nucleotides/day (9000 AP sites/cell/day). This highly sensitive assay allows us to determine the endogenous level of AP sites in genomic DNA, as well as to investigate whether DNA-damaging agents cause imbalances of base excision/AP endonuclease repair in vivo and in vitro.

https://cancerres.aacrjournals.org/content/canres/58/2/222.full.pdf

Highly sensitive apurinic/apyrimidinic site assay can detect spontaneous and chemically induced depurination under physiological conditions

Oxidative DNA damage repair in mammalian cells: a new perspective.

Thymine glycols were produced in M13 DNA in a concentration dependent manner by treating the DNA with osmium tetroxide (OsO4). For the formation of urea-containing M13 DNA, OsO4-oxidized DNA was hydrolyzed in alkali (pH 12) to convert the thymine glycols to urea residues. With both thymine glycol- and urea-containing M13 DNA, DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment was decreased in proportion to the number of damages present in the template DNA. Sequencing gel analysis of the products synthesized by E. coli DNA polymerase I and T4 DNA polymerase showed that DNA synthesis terminated opposite the putative thymine glycol site and at one nucleotide before the putative urea site. Substitution of manganese for magnesium in the reaction mix resulted in increased processivity of DNA synthesis so that a base was incorporated opposite urea. With thymine glycol-containing DNA, processivity in the presence of manganese was strongly dependent on the presence of a pyrimidine 5' to the thymine glycol in the template.

/pdf/thymine-glycols-and-urea-residues-in-m13-dna-constitute-598u73lmio.pdf

Yoke W. Kow

Papers

Identification and characterization of a human DNA glycosylase for repair of modified bases in oxidatively damaged DNA.

Identification and characterization of a novel human DNA glycosylase for repair of cytosine-derived lesions.

Highly sensitive apurinic/apyrimidinic site assay can detect spontaneous and chemically induced depurination under physiological conditions

Oxidative DNA damage repair in mammalian cells: a new perspective.

Thymine glycols and urea residues in M13 DNA constitute replicative blocks in vitro