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DNA Damage and Repair

Minor Bases for DNA Damage and Repair

Cellular DNA is constantly being damaged by oxidation and alkylation, by free radicals, and by ultraviolet and ionizing radiation. The body has therefore evolved a number of repair enzyme systems to excise and repair these lesions. The 8-oxo purine monomers allow investigation of the structure and activity of oligonucleotides containing an 8-oxo mutation which is formed naturally when DNA is subjected to oxidative conditions or ionizing radiation. 5,6-Dihydro pyrimidines are naturally occurring compounds that are structural components of alanine transfer RNA. Dihydrouracil and the hydroxy pyrimidines are major base damage products formed by exposure of DNA to ionizing radiation.

  • 8-Oxo dA
  • 8-Oxo dG
  • 5-OH dC
  • 5-OH dU
  • 5-Hydroxymethyl dU
  • 5,6 Dihydro dT
  • 1-Methyl dA
  • N3-Cyanoethyl dT
  • 5,6 Dihydro dU
  • Pyrene dU
  • Perylene dU

8-Amino-G is formed along with 8-oxo-G as the major mutagenic lesions formed in DNA damage caused by 2-nitropropane. 2-Nitropropane is an industrial solvent and a component of paints, dyes and varnishes, and is also present in cigarette smoke. Thymine glycol (5,6-dihydroxy-5,6-dihydrothymine) is formed when thymine is subjected to oxidative stress, including ionizing radiation. Oxidation of the 5,6 double bond of Thymidine generates two chiral centers at C5 and C6. The cis-5R,6S form is generated as the predominant product along with the other diastereomer, the cis-5S,6R form. The presence of thymidine glycol in DNA has significant biological consequences and many organisms possess specific repair enzymes for the excision of this lesion. 2-Aminoimidazolone (Iz) and its hydrolysis product imidazolone (Z) are major oxidation products of G. Access to these two potential lesions is not possible during oligonucleotide synthesis because they are so base-labile. A suitable precursor, 8-methoxy-dG (8-OMe-dG), to dIz has now been described. The conversion of 8-OMe-dG to dIz takes place by irradiation of the oligonucleotide (1 mM) in 50 mM sodium cacodylate buffer, pH 7, in the presence of riboflavin (50 µM) for 2 minutes on a transilluminator (366 nm), under aerobic conditions at 4°C. Surprisingly for a photochemical reaction, the conversion is virtually quantitative. Hydrolysis of nucleoside residues in DNA occurs naturally to generate abasic sites. Most commonly, dA sites are hydrolyzed causing depurination and leading to abasic residues. A new chemical method allows the generation of abasic sites in double and single stranded oligonucleotides using very mild specific conditions and with very low probability of side reactions. dSpacer has also been used successfully as a mimic of the highly base-labile abasic site.

  • 8-Amino dG
  • 8-OMe dG
  • Thymidine Glyco
  • Abasic

One of the major sources of DNA damage in all organisms is the UV component of sunlight. The predominant reaction induced by UV light on DNA is dimerization of adjacent pyrimidine bases leading to cyclobutane dimers (CPDs). The dimers formed in the most significant quantity are the cis-syn cyclobutane dimer of two thymine bases. Although formed routinely, these dimer products are efficiently excised and repaired enzymatically (nucleotide excision repair) or the dimerization is reversed by photolase enzymes. These lesions have been connected to the formation of squamous cell carcinomas. In addition, humans who lack ability to repair CPD lesions with high efficiency may be genetically predisposed to Xeroderma Pigmentosa (XP), a disease characterized by extreme sensitivity to sunlight and high frequency of skin cancer. Polymerases encountering unrepaired CPD lesions are quite error-prone, leading to incorrect base insertions and subsequent mutations.

  • Cis-syn Thymine Dimer

One of the most studied repair mechanisms is probably the base excision DNA repair pathway. In this pathway, DNA glycosylases recognize the damaged bases and catalyze their excision through hydrolysis of the N-glycosidic bond. Attempts to understand the structural basis for DNA damage recognition by DNA glycosylases have been hampered by the short-lived association of these enzymes with their DNA substrates. To overcome this problem, the design and synthesis of inhibitors that form stable complexes with DNA glycosylases are essential. Complexes can then be studied biochemically and structurally. Toward this end, the Verdine group at Harvard synthesized a pyrrolidine analog that mimics the charged transition state of the enzyme-substrate complex. When incorporated into double-stranded DNA, they found the pyrrolidine analog (PYR), forms an extremely stable complex with the DNA glycosylase AlkA, exhibiting a dissociation constant in the pM range and potently inhibited the reaction catalyzed by the enzyme.

  • Pyrrolidine