Hypoxia induces mitochondrial DNA damage and stimulates expression of a DNA repair enzyme, the Escherichia coli MutY DNA glycosylase homolog (MYH), in vivo, in the rat brain

Hypoxia-associated, acutely reduced blood oxygenation can compromise energy metabolism, alter oxidant/antioxidant balance and damage cellular components, including DNA. We show in vivo, in the rat brain that respiratory hypoxia leads to formation of the oxidative DNA lesion, 8-hydroxy-2'-deoxyguanosine (oh8dG), a biomarker for oxidative DNA damage and to increased expression of a DNA repair enzyme involved in protection of the genome from the mutagenic consequences of oh8dG. The enzyme is a homolog of the Escherichia coli MutY DNA glycosylase (MYH), which excises adenine residues misincorporated opposite the oxidized base, oh8dG. We have cloned a full-length rat MYH (rMYH) cDNA, which encodes 516 amino acids, and by in situ hybridization analysis obtained expression patterns of rMYH mRNA in hippocampal, cortical and cerebellar regions. Ensuing hypoxia, mitochondrial DNA damage was induced and rMYH expression strongly elevated. This is the first evidence for a regulated expression of a DNA repair enzyme in the context of respiratory hypoxia. Our findings support the premise that oxidative DNA damage is repaired in neurons and the possibility that the hypoxia-induced expression of a DNA repair enzyme in the brain represents an adaptive mechanism for protection of neuronal DNA from injurious consequences of disrupted energy metabolism and oxidant/antioxidant homeostasis.     

Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences

The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase β (POL β) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL β incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL β was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences.     

Endogenously generated DNA nucleobase modifications source,and significance as possible biomarkers of malignant transformation risk,and role in anticancer therapy

The DNA of all living cells undergoes continuous structural and chemical alteration, which may be derived from exogenous sources, or endogenous, metabolic pathways, such as cellular respiration, replication and DNA demethylation. It has been estimated that approximately 70,000 DNA lesions may be generated per day in a single cell, and this has been linked to a wide variety of diseases, including cancer. However, it is puzzling why potentially mutagenic DNA modifications, occurring at a similar level in different organs/tissue, may lead to organ/tissue specific cancers, or indeed non-malignant disease – what is the basis for this differential response? We suggest that it is perhaps the precise location of damage, within the genome, that is a key factor. Finally, we draw attention to the requirement for reliable methods for identification and quantification of DNA adducts/modifications, and stress the need for these assays to be fully validated. Once these prerequisites are satisfied, measurement of DNA modifications may be helpful as a clinical parameter for treatment monitoring, risk group identification and development of prevention strategies.     

Role of 3-methyladenine-DNA glycosylase in host-cell reactivation of methylated T7 bacteriophage

Purified T7 phage, treated with methyl methanesulfonate, was assayed on four Escherichia coli K12 host cells: (1) AB1157, wild-type; (2) PK432-1, lacking 3-methyladenine-DNA glycosylase (tag); (3) NH5016, lacking apurinic endonuclease VI (xthA); (4) p3478, lacking DNA polymerase I (polA), the latter three strains being deficient in enzymes of the base excision repair pathway. For inactivation measured immediately after alkylation, phage survival was lowest on strains PK432-1 and p3478; for delayed inactivation, measured after partial depurination of alkylated phage, survival was much lower on strain p3478 than on PK432-1. These results demonstrate the important role played by 3-methyladenine-DNA glycosylase in the survival of methylated T7 phage. Quantitative analysis of the data, using the results of Verly et al. (Verly, W.G., Crine, P., Bannon, P. and Forget, A. (1974) Biochim. Biophys. Acta 349, 204–213) to correlate the dose with the number of methyl groups introduced into phage DNA, revealed that 5–10 3-methyladenine residues per T7 DNA constituted an inactivation hit for the tag mutant. Thus, 3-methyladenine may be as toxic a lesion as an apurinic site.     

Plausible biochemical mechanisms of chemotherapy-induced cognitive impairment (“chemobrain”), a condition that significantly impairs the quality of life of many cancer survivors

Increasing numbers of cancer patients survive and live longer than five years after therapy, but very often side effects of cancer treatment arise at same time. One of the side effects, chemotherapy-induced cognitive impairment (CICI), also called “chemobrain” or “chemofog” by patients, brings enormous challenges to cancer survivors following successful chemotherapeutic treatment. Decreased abilities of learning, memory, attention, executive function and processing speed in cancer survivors with CICI, are some of the challenges that greatly impair survivors' quality of life. The molecular mechanisms of CICI involve very complicated processes, which have been the subject of investigation over the past decades. Many mechanistic candidates have been studied including disruption of the blood-brain barrier (BBB), DNA damage, telomere shortening, oxidative stress and associated inflammatory response, gene polymorphism of neural repair, altered neurotransmission, and hormone changes. Oxidative stress is considered as a vital mechanism, since over 50% of FDA-approved anti-cancer drugs can generate reactive oxygen species (ROS) or reactive nitrogen species (RNS), which lead to neuronal death. In this review paper, we discuss these important candidate mechanisms, in particular oxidative stress and the cytokine, TNF-alpha and their potential roles in CICI.     

Correlated cleavage of single- and double-stranded substrates by uracil-DNA glycosylase

Uracil-DNA glycosylase (Ung) can quickly locate uracil bases in an excess of undamaged DNA. DNA glycosylases may use diffusion along DNA to facilitate lesion search, resulting in processivity, the ability of glycosylases to excise closely spaced lesions without dissociating from DNA. We propose a new assay for correlated cleavage and analyze the processivity of Ung. Ung conducted correlated cleavage on double- and single-stranded substrates; the correlation declined with increasing salt concentration. Proteins in cell extracts also decreased Ung processivity. The correlated cleavage was reduced by nicks in DNA, suggesting the intact phosphodiester backbone is important for Ung processivity.     

Structure and mechanism for DNA lesion recognition

A fundamental question in DNA repair is how a lesion is detected when embedded in millions to billions of normal base pairs. Extensive structural and functional studies reveal atomic details of DNA repair protein and nucleic acid interactions. This review summarizes seemingly diverse structural motifs used in lesion recognition and suggests a general mechanism to recognize DNA lesion by the poor base stacking. After initial recognition of this shared structural feature of lesions, different DNA repair pathways use unique verification mechanisms to ensure correct lesion identification and removal.     

Co-transcription of a homologue of the formamidopyrimidine-DNA glycosylase (fpg) and lysophosphatidic acid acyltransferase (nlaA) in Neisseria meningitidis

Abstract We report the identification of an open reading frame in a serogroup B isolate of Neisseria meningitidis that exhibits high nucleotide and predicted amino acid identity with the fpg gene of Escherichia coli , and its product, formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosylase), a DNA repair enzyme. We further show that the meningococcal fpg is co-transcribed with nlaA , encoding a lysophosphatidic acid acyltransferase, and suggest that the DNA repair enzyme may be involved in the regulation of nlaA or its gene product.