Bulky DNA Adducts

Therefore, the formation of alkyl DNA or bulky DNA adducts can be regarded as structural modifications of DNA and so are potentially premutagenic lesions. 

The nucleotide excision repair (NER) pathway is involved in the removal of a wide range of bulky DNA adducts and DNA cross-hnks. In addition, the NER enzymes may recognize many diverse DNA lesions that do not disturb the DNA helix, including 8-oxodG and other modified bases. Under normal conditions NER does not contribute significantly (maybe as httle as 0.1% [39]) to the removal of 8-oxodG in mammahan cell extracts, not even when the pol j8-de-pendent BER pathway was disrupted [33]. It is, however, a common assumption that NER works as a back up system for BER, i.e., when BER is deficient NER can repair the base modifications. 

Specific DNA adducts are excreted into urine as modified bases and deoxynucleotides [121-123]. As endonucleases and glycosylases repair the damaged DNA, deoxynucleotides and bases respectively are liberated. Oligonucleotides and bulky DNA adducts like benzo[a]pyrene-deoxynucleotides or aflatoxin-Bj-Ny-dG are also excreted. Immuno-affinity chromatography, GC/MS, LC-MS/MS,or HPLE-EC can be used to quantify the wide range of base modifications and thus to estimate the DNA repair activity, whereas ELISA assays do not yet have sufficient proven specificity. 

Cai, Y., Patel, D.J., Geadntov, N.E., and Broyde, S. (2009) Differential nucleotide exdsion repair susceptibility of bulky DNA adducts in different sequence contexts hierarchies of recognition signals./. Mol. Biol., 385, 304-4. 

Denissenko, M.F., Pao, A., Pfeifer, G.P., and Tang, M.-S. (1998) Slow repair of bulky DNA adducts along the nontran-scribed strand of the human p53 gene may explain the strand bias of... 

We have isolated these domains from hepatocytes in different stages of DNA excision repair (2, 3) and studied the distribution of newly synthesized repair patches as well as the position of bulky DNA adducts in Aese sites. The advantage of this approach as compared to micrococcal nuclease probing of chromatin is that 8-methoxypsoralen can be covalently bound to free DNA domains in situ, and subsequent ffactionation of chromatin does not effect its position (2-7). 

Akcha et al. (2000) M. galloprovincialis Exposure to 1.786 ig BaP/ mg dw feed for 28 days (total experiment duration) BPH, CAT, DTD (digestive gland) Two distinct bulky DNA adducts in digestive gland... 

There is strong evidence that DNA adduction by these bulky reactive metabolites of PAHs is far from random, and that there are certain hot spots that are preferentially attacked. Differential steric hindrance and the differential operation of DNA repair mechanisms ensure that particular sites on DNA are subject to stable adduct formation (Purchase 1994). DNA repair mechanisms clearly remove many PAH/ guanine adducts very quickly, but studies with P postlabeling have shown that certain adducts can be very persistent—certainly over many weeks. Evidence for this has been produced in studies on fish and Xenopus (an amphibian Reichert et al. 1991 Waters et al. 1994). 

Reichert, W.L., French, B.L, and Stein, J.E. et al. (1991). 32P postlabelling analysis of the persistence of bulky hydrophobic xenobiotic-DNA adducts in the liver of English sole, a marine fish. In Proceedings of the 82nd Meeting of the American Association for Cancer Research, Houston, TXP, 87. 

If a DNA adduct involves the nitrogen or oxygen atoms involved in base-pairing, and the adducted DNA is not repaired, base substitution can result. Adducts can be small, such as the simple addition of methyl or ethyl groups, or they can be very bulky, owing to reaction with multiringed structures. The most vulnerable base is guanine, which can form adducts at several of its atoms (e.g., N7, C8, O6 and exocyclic N2) (Venitt and Parry, 1984). Adducts can form links between adjacent bases on the same strand (intrastrand cross-links) and can form interstrand crosslinks between each strand of double-stranded DNA. 

Exposure of cells to carcinogens may result in the formation of DNA adducts varying in size from methyl groups to bulky structures, such as metabolites of polycyclic aromatic hydrocarbons and aromatic amines. In vivo, these adducts usually are removed enzymatically and at different rates from the DNA. Because the liver is the main site of activation of chemical carcinogens, the DNA of this organ usually forms more adducts. Direct detection and measurement of DNA damage are thus possible, in principle, by detection and measurement of the bound adduct. Because the number of adducts is usually extremely small, very sensitive methods cure required for their measurement. 

Benzo[n]pyrene is an extensively studied PAH compound. The mutagenic metabolites of benzo[a]pyrene are the (+)-7R,85,95,101 -flm/-benzo[fl]pyrene-7,8-dihydrodiol-9,10-epoxide, (+)-a //-BPDE, and the (-)-7R,8S,9S,10R enantiomer, ( )-anti-BPDE (Figure 22.16A,B). DNA damage occurs mainly by adduct formation between the CIO position of anti-BPDE to the N2 position of guanine. Four stereoisomeric bulky adducts are produced 105 (+)-trans-anti-BPDE-N2-dG, 1 OR (+)-cis-anti-BPE>E-N2-dG, 1 OR (-)-trans-anti-BPDE-N2-dG, and 105 (-)-cis-anti-BPDE-A2-dG. In vitro, the reaction of (+)-anti-BPDE with DNA forms predominantly the (+)-trans-anti-BPE)E-N2-dG adduct, while the reaction of ( )-anti-BPDE produces mainly the ( )-trans-anti-BPDE-N2-dG adduct. In cells, the major benzo[a]pyrene DNA adduct is (+)-trans-anti-BPDE-N2-dG. 

When benzo(a)pyrene (a carcinogen in cigarette smoke) binds to DNA, it forms a bulky covalent adduct on guanine residues. The consequence is that... 

DNA primary damage DNA adducts (alkyl and bulky adducts) are nucleotide bases covalently modified by reactive electrophilic chemical intermediates or free radicals. 

Purpose. DNA adducts are nucleotide bases (i.e., purines and pyrimidines) that have been covalently modified by reactive electrophilic chemical intermediates or free radicals. The chemical structures of DNA adducts are diverse and vary from simple alkyl adducts induced by alkylating agents to complex bulky adducts such as those resulting from metabolic activation of polycyclic aromatic hydrocarbons, aromatic amines, and aflatoxins (Dipple 1995 Chiarelli and Jackson 1992 Rundle 2006 Xue and Warshawsky 2005). The purpose of measuring DNA adducts is to determine whether a DNA-reactive compound or a metabolically activated... 

TFIIH also partidpates in another important cellular function, namely nucleotide excision repair of damaged DNA. This function accounts for the observation that transcription and the removal of bulky base adducts by nucleotide excision repair (NER) are coupled. An increased repair of DNA damage by NER is observed while a gene is being transcribed. During transcription-coupled repair, TFIIH assembles with other repair proteins into a large repair complex, allowing for the removal of DNA adducts. 

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