Amanda K. McCullough

Cellular pathways for DNA repair and damage tolerance of formaldehyde-induced DNA-protein crosslinks.

Although it is well established that DNA-protein crosslinks are formed as a consequence of cellular exposure to agents such as formaldehyde, transplatin, ionizing and ultraviolet radiation, the biochemical pathways that promote cellular survival via repair or tolerance of these lesions are poorly understood. To investigate the mechanisms that function to limit DNA-protein crosslink-induced cytotoxicity, the Saccharomyces cerevisiae non-essential gene deletion library was screened for increased sensitivity to formaldehyde exposure. Following low dose, chronic exposure, strains containing deletions in genes mediating homologous recombination showed the greatest sensitivity, while under the same exposure conditions, deletions in genes associated with nucleotide excision repair conferred only low to moderate sensitivities. However, when the exposure regime was changed to a high dose acute (short-term) formaldehyde treatment, the genes that conferred maximal survival switched to the nucleotide excision repair pathway, with little contribution of the homologous recombination genes. Data are presented which suggest that following acute formaldehyde exposure, repair and/or tolerance of DNA-protein crosslinks proceeds via formation of nucleotide excision repair-dependent single-strand break intermediates and without a detectable accumulation of double-strand breaks. These data clearly demonstrate a differential pathway response to chronic versus acute formaldehyde exposures and may have significance and implications for risk extrapolation in human exposure studies.

Human Polymorphic Variants of the NEIL1 DNA Glycosylase

In mammalian cells, the repair of DNA bases that have been damaged by reactive oxygen species is primarily initiated by a series of DNA glycosylases that include OGG1, NTH1, NEIL1, and NEIL2. To explore the functional significance of NEIL1, we recently reported that neil1 knock-out and heterozygotic mice develop the majority of symptoms of metabolic syndrome (Vartanian, V., Lowell, B., Minko, I. G., Wood, T. G., Ceci, J. D., George, S., Ballinger, S. W., Corless, C. L., McCullough, A. K., and Lloyd, R. S. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1864-1869). To determine whether this phenotype could be causally related to human disease susceptibility, we have characterized four polymorphic variants of human NEIL1. Although three of the variants (S82C, G83D, and D252N) retained near wild type levels of nicking activity on abasic (AP) site-containing DNA, G83D did not catalyze the wild type β,δ-elimination reaction but primarily yielded the β-elimination product. The AP nicking activity of the C136R variant was significantly reduced. Glycosylase nicking activities were measured on both thymine glycol-containing oligonucleotides and γ-irradiated genomic DNA using gas chromatography/mass spectrometry. Two of the polymorphic variants (S82C and D252N) showed near wild type enzyme specificity and kinetics, whereas G83D was devoid of glycosylase activity. Although insufficient quantities of C136R could be obtained to carry out gas chromatography/mass spectrometry analyses, this variant was also devoid of the ability to incise thymine glycol-containing oligonucleotide, suggesting that it may also be glycosylase-deficient. Extrapolation of these data suggests that individuals who are heterozygous for these inactive variant neil1 alleles may be at increased risk for metabolic syndrome.

Characterization of a novel cis-syn and trans-syn-II pyrimidine dimer glycosylase/AP lyase from a eukaryotic algal virus, Paramecium bursaria chlorella virus-1

Endonuclease V from bacteriophage T4, is acis-syn pyrimidine dimer-specific glycosylase. Recently, the first sequence homolog of T4 endonuclease V was identified from chlorella virus Paramecium bursaria chlorella virus-1 (PBCV-1). Here we present the biochemical characterization of the chlorella virus pyrimidine dimer glycosylase, cv-PDG. Interestingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer. This is the first trans-syn-II-specific glycosylase identified to date. Kinetic analysis demonstrates that DNAs containing both types of pyrimidine dimers are cleaved by the enzyme with similar catalytic efficiencies. Cleavage analysis and covalent trapping experiments demonstrate that the enzyme mechanism is consistent with the model proposed for glycosylase/AP lyase enzymes in which the glycosylase action is mediated via an imino intermediate between the C1′ of the sugar and an amino group in the enzyme, followed by a β-elimination reaction resulting in cleavage of the phosphodiester bond. cv-PDG exhibits processive cleavage kinetics which are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic attraction between enzyme and DNA. The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endonuclease V-like enzymes with specificity toward other DNA photoproducts.

The role of base flipping in damage recognition and catalysis by T4 endonuclease V

The process of moving a DNA base extrahelical (base flipping) has been shown in the co-crystal structure of a UV-induced pyrimidine dimer-specific glycosylase, T4 endonuclease V, with its substrate DNA. Compared with other enzymes known to use base flipping, endonuclease V is unique in that it moves the base opposite the target site extrahelical, rather than moving the target base itself. Utilizing substrate analogs and catalytically inactive mutants of T4 endonuclease V, this study investigates the discrete steps involved in damage recognition by this DNA repair enzyme. Specifically, fluorescence spectroscopy analysis shows that fluorescence changes attributable to base flipping are specific for only the base directly opposite either abasic site analogs or the 5′-thymine of a pyrimidine dimer, and no changes are detected if the 2-aminopurine is moved opposite the 3′-thymine of the pyrimidine dimer. Interestingly, base flipping is not detectable with every specific binding event suggesting that damage recognition can be achieved without base flipping. Thus, base flipping does not add to the stability of the specific enzyme-DNA complex but rather induces a conformational change to facilitate catalysis at the appropriate target site. When used in conjunction with structural information, these types of analyses can yield detailed mechanistic models and critical amino acid residues for extrahelical base movement as a mode of damage recognition.

The reaction mechanism of DNA glycosylase/AP lyases at abasic sites.

DNA glycosylase and glycosylase/abasic (AP) lyases are the enzymes responsible for initiating the base excision repair pathway by recognizing the damaged target base and catalyzing the breakage of the base-sugar glycosyl bond. The subset of glycosylases that have an associated AP lyase activity also catalyze DNA strand breakage at the resulting or preexisting AP site via a beta-elimination reaction, proceeding from an enzyme-DNA imino intermediate. Two distinct mechanisms have been proposed for the formation of this intermediate. These mechanisms essentially differ in the nature of the first bond broken and the timing of the opening of the deoxyribose ring. The data presented here demonstrate that the combined rate of sugar ring opening and reduction of the sugar is significantly slower than the rate of formation of a T4-pyrimidine dimer glycosylase (T4-pdg)-DNA intermediate. Using a methyl-deoxyribofuranose AP-site analogue that is incapable of undergoing sugar ring opening, it was demonstrated that the T4-pdg reaction can initiate at the ring-closed form, albeit at a drastically reduced rate. T4-pdg preferentially cleaved the beta-anomer of the methyl-deoxyribofuranose AP site analogue. This is consistent with a mechanism in which the methoxy group is backside-displaced by the amino group from the alpha-face of the deoxyribofuranose ring. In addition, studies examining rates of sugar-aldehyde reduction and the sodium borohydride concentration dependence of the rate of formation of the covalent imine intermediate suggest that the reduction of the intermediate is rate-limiting in the reaction.

Regulation of DNA glycosylases and their role in limiting disease

Abstract This review will present a current understanding of mechanisms for the initiation of base excision repair (BER) of oxidatively-induced DNA damage and the biological consequences of deficiencies in these enzymes in mouse model systems and human populations.

Formaldehyde-Induced Genome Instability is Suppressed by an XPF-dependent Pathway

Formaldehyde is a reactive chemical that is commonly used in the production of industrial, laboratory, household, and cosmetic products. The causal association between formaldehyde exposure and increased incidence of cancer led the International Agency for Research on Cancer to classify formaldehyde as a carcinogen. Formaldehyde-induced DNA-protein crosslinks (DPCs) elicit responses involving nucleotide excision repair (NER) and homologous recombination (HR) repair pathways; however, little is known about the cellular and genetic changes that subsequently lead to formaldehyde-induced genotoxic and cytotoxic effects. Herein, investigations of genes that modulate the cytotoxic effects of formaldehyde exposure revealed that of five NER-deficient Chinese Hamster Ovary (CHO) cell lines tested, XPF- and ERCC1-deficient cells were most sensitive to formaldehyde treatment as compared to wild-type cells. Cell cycle analyses revealed that formaldehyde-treated XPF-deficient cells exhibited an immediate G2/M arrest that was associated with altered cell ploidy and apoptosis. Additionally, an elevated number of DNA double-strand breaks (DSBs), chromosomal breaks and radial formation were also observed in XPF-deficient cells following formaldehyde treatment. Formaldehyde-induced DSBs occurred in a replication-dependent, but an XPF-independent manner. However, delayed DSB repair was observed in the absence of XPF function. Collectively, our findings highlight the role of an XPF-dependent pathway in mitigating the sensitivity to formaldehyde-induced DNA damage as evidenced by the increased genomic instability and reduced cell viability in an XPF-deficient background. In addition, centrosome and microtubule abnormalities, as well as enlarged nuclei, caused by formaldehyde exposure are demonstrated in a repair-proficient cell line.

Variable penetrance of metabolic phenotypes and development of high-fat diet-induced adiposity in NEIL1-deficient mice

Exposure to chronic and acute oxidative stress is correlated with many human diseases, including, but not limited to, cancer, heart disease, diabetes, and obesity. In addition to cellular lipids and proteins, cellular oxidative stress can result in damage to DNA bases, especially in mitochondrial DNA. We previously described the development of spontaneous late-onset obesity, hepatic steatosis, hyperinsulinemia, and hyperleptinemia in mice that are deficient in the DNA glycosylase nei-like 1 (NEIL1), which initiates base excision repair of several oxidatively damaged bases. In the current study, we report that exposure to a chronic oxidative stress in the form of a high-fat diet greatly accelerates the development of obesity in neil1(-/-) mice. Following a 5-wk high-fat diet challenge, neil1(-/-) mice gained significantly more body weight than neil1(+/+) littermates and had increased body fat accumulation and moderate to severe hepatic steatosis. Analysis of oxygen consumption by indirect calorimetry indicated a modest reduction in total oxygen consumption in neil1(-/-) mice that was abolished upon correction for lean body mass. Additionally, hepatic expression of several inflammatory genes was significantly upregulated in neil1(-/-) mice following high-fat diet challenge compared with chow-fed or neil1(+/+) counterparts. A long-term high-fat diet also induced glucose intolerance as well as a significant reduction in mitochondrial DNA and protein content in neil1(-/-) mice. Collectively, these data indicate that NEIL1 deficiency results in an increased susceptibility to obesity and related complications potentially by lowering the threshold for tolerance of cellular oxidative stress in neil1(-/-) mice.

Structural Determinants for Specific Recognition by T4 Endonuclease V

DNA glycosylases catalyze the scission of the N-glycosyl bond linking either a damaged or mismatched base to the DNA sugar phosphate backbone. T4 endonuclease V is a glycosylase/apurinic (AP) lyase that is specific for UV light-induced cis-syn pyrimidine dimers. As a proposed transition state analog/inhibitor for glycosylases, a phosphoramidite derivative containing a pyrrolidine residue has been synthesized. The binding of endonuclease V to this duplex was analyzed by gel mobility shift assays and resulted in a single stable complex of reduced mobility and an apparent Kd of 17 nM. To assess the importance of the positive charge for specific binding, studies using other non-cleavable substrate analogs were performed. Wild type T4 endonuclease V shows an 8-fold decreased affinity for a tetrahydrofuran as compared with the pyrrolidine residue, demonstrating the significance of the positive charge for recognition. A 2-fold increase in binding affinity for a reduced AP site was observed. Similar assays using catalytically compromised mutants (E23Q and E23D) of endonuclease V demonstrate altered affinities for the pyrrolidine as well as tetrahydrofuran and reduced AP sites. This approach has provided insight into the structural mechanism by which specific lesions are targeted by the protein as well as the structural determinants of the DNA required for specific recognition by T4 endonuclease V.

Modulation of UvrD Helicase Activity by Covalent DNA-Protein Cross-links

UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3′ to 5′ direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD interactions with proteins bound to DNA have significant biological implications, the effects of covalent DNA-protein cross-links on UvrD helicase activity have not been characterized. Herein, we demonstrate that UvrD-catalyzed strand separation was inhibited on a DNA strand to which a 16-kDa protein was covalently bound. Our sequestration studies suggest that the inhibition of UvrD activity is most likely due to a translocation block and not helicase sequestration on the cross-link-containing DNA substrate. In contrast, no inhibition of UvrD-catalyzed strand separation was apparent when the protein was linked to the complementary strand. The latter result is surprising given the earlier observations that the DNA in this covalent complex is severely bent (∼70°), with both DNA strands making multiple contacts with the cross-linked protein. In addition, UvrD was shown to be required for replication of plasmid DNAs containing covalent DNA-protein complexes. Combined, these data suggest a critical role for UvrD in the processing of DNA-protein cross-links.