UV photodissociation dynamics of deprotonated 2'-deoxyadenosine 5'-monophosphate [5'-dAMP-H]-.

The UV photodissociation dynamics of deprotonated 2'-deoxyadenosine 5'-monophosphate ([5'-dAMP-H](-)) has been studied using a unique technique based on the coincident detection of the ion and the neutral fragments. The observed fragment ions are m/z 79 (PO(3)(-)), 97 (H(2)PO(4)(-)), 134 ([A-H](-)), 177 ([dAMP-H-A-H(2)O](-)), and 195 ([dAMP-H-A](-)), where "A" refers to a neutral adenine molecule. The relative abundances are comparable to that found in previous studies on [5'-dAMP-H](-) employing different excitation processes, i.e., collisions and UV photons. The fragmentation times of the major channels have been measured, and are all found to be on the microsecond time scale. The fragmentation mechanisms for all channels have been characterized using velocity correlation plots of the ion and neutral fragment(s). The findings show that none of the dissociation channels of [5'-dAMP-H](-) is UV specific and all proceed via statistical fragmentation on the ground state after internal conversion, a result similar to fragmentations induced by collisions.

Termination of DNA synthesis by N6-alkylated, not 3'-O-alkylated, photocleavable 2'-deoxyadenosine triphosphates.

The Human Genome Project has facilitated the sequencing of many species, yet the current Sanger method is too expensive, labor intensive and time consuming to accomplish medical resequencing of human genomes en masse. Of the 'next-generation' technologies, cyclic reversible termination (CRT) is a promising method with the goal of producing accurate sequence information at a fraction of the cost and effort. The foundation of this approach is the reversible terminator (RT), its chemical and biological properties of which directly impact the performance of the sequencing technology. Here, we have discovered a novel paradigm in RT chemistry, the attachment of a photocleavable, 2-nitrobenzyl group to the N(6)-position of 2'-deoxyadenosine triphosphate (dATP), which, upon incorporation, terminates DNA synthesis. The 3'-OH group of the N(6)-(2-nitrobenzyl)-dATP remains unblocked, providing favorable incorporation and termination properties for several commercially available DNA polymerases while maintaining good discrimination against mismatch incorporations. Upon removal of the 2-nitrobenzyl group with UV light, the natural nucleotide is restored without molecular scarring. A five-base experiment, illustrating the exquisite, stepwise addition through a homopolymer repeat, demonstrates the applicability of the N(6)-(2-nitrobenzyl)-dATP as an ideal RT for CRT sequencing.

Characterization of the reactive intermediates in laser flash photolysis of adenine, adenosine and dAMP using acetone as photosensitizer.

Transient absorption spectra of adenine, adenosine and 2'-deoxyadenosine 5'-monophosphate (dAMP) arising from 248 nm laser flash photolysis using acetone as a photosensitizer have been observed. The intermediates recorded are assigned to the excited triplet states and dehydrogenated radicals of adenine and its nucleoside and nucleotide. The excited triplet states of adenine and its derivatives are produced via triplet-triplet excitation transfer and observed for the first time, while the dehydrogenated radicals stemming from the interaction of triplet acetone with adenine and its derivatives via electron transfer through a five-member-ring electron donor-acceptor intermediate. The site of dehydrogenation is suggested to be the hydrogen atom on C(8) of the adenine moiety. Moreover, three sets of kinetic parameters of the triplet decay have been determined. The rate constants of the unimolecular decay (k0), the triplet quenching by the ground state (ksq) and by the triplet quencher Mn2+ (kq) are 1.1 x 10(5), 7.9 x 10(4), 3.7 x 10(4) s-1, 6.9 x 10(8), 8.3 x 10(8), 3.6 x 10(8) dm3 mol-1 s-1 and 4.2 x 10(8), 3.5 x 10(8), 6.0 x 10(8) dm3 mol-1 s-1 respectively for adenine, adenosine and dAMP.

Reaction of cysteamine with individual DNA base radicals in gamma-irradiated nucleotides at low temperature.

An ESR investigation of the individual DNA base radicals produced by gamma-irradiation of frozen solutions of the nucleotides TMP, dCMP, dGMP and dAMP and their reactions with cysteamine upon annealing is reported. The results show that water radicals in bulk ice do not lead to the formation of DNA or cysteamine radicals. Radicals from the oxidation pathway which include the DNA base one electron oxidized radicals and their successors, G(C8)OH., A(C8)OH. and thymine dimers (.Tdi) and/or T(C6)OH; readily react with cysteamine to form RS. and ultimately RSSR-. Reactions of dGMP and dCMP radicals from the oxidation pathway with cysteamine occur at lower temperatures than those of dAMP and TMP, suggesting hole migration. Both T(C6)H. and C(N3)H. react with cysteamine to form RS. and diamagnetic products, but G(C8)H. and A(C8)H. do not. Subtraction of the anion radical T-. and its proton adduct T(C6)H. from the total radical yield of TMP (with or without cysteamine) suggests that somewhat less than half of the total TMP radicals found are a result of the oxidative pathway. Similar results are found in the other nucleotides. The total spectral intensity derived from the radicals from the oxidative pathway such as G(C8)OH., A(C8)OH. and .Tdi/T(C6)OH. are somewhat less than that for the protonated anion radicals. Only one non-base radical is identified, a sugar radical at the C(1)' site on the deoxyribose portion of dAMP. This species, S(A)., is also found to react with cysteamine or its disulfide radical anion. Analyses performed in the presence and absence of a thiol are found to allow for a clear separation of oxidative and reductive pathways.

The isolation and characterisation of a new type of dimeric adenine photoproduct in UV-irradiated deoxyadenylates.

A new type of dimeric adenine photoproduct has been isolated from d(ApA) irradiated at 254 nm in neutral aqueous solution. It is formed in comparable amounts to another, quite distinct, adenine photoproduct first described by Pörschke (J. Am. Chem. Soc. (1973), 95, 8440-8446). Results from high resolution mass spectrometry and 1H NMR indicate that the new photoproduct comprises a mixture of two stereoisomers whose formation involves covalent coupling of the adenine bases in d(ApA) and concomitant incorporation of the elements of one molecule of water. The photoproduct is degraded specifically by acid to 4,6-diamino-5-guanidinopyrimidine (DGPY) whose identity has been confirmed by independent chemical synthesis. Formation of the new photoproduct in UV-irradiated d(pA)2 and poly(dA), but not poly(rA), has been demonstrated by assaying their acid hydrolysates for the presence of DGPY. The properties of the photoproduct are consistent with it being generated by the hydrolytic fission of an azetidine photoadduct in which the N(7) and C(8) atoms of the 5'-adenine in d(ApA) are linked respectively to the C(6) and C(5) positions of the 3'-adenine.

DNA radiation damage and its modification by metallothionein.

Thiol compounds have long been known to protect living cells against the harmful effects of ionizing radiation. Maetallothionein is a naturally occurring low molecular weight polypeptide rich in cysteine residues and may be useful in protection against low-level radiation effects. Radiation damage to DNA and its nucleotide components and the radioprotective effect of metallothionein have been studied in model chemical systems and compared to its effect on cells. Metallothionein acts both as a free radical scavenger and a reductant, and its radioprotective effectiveness has been studied as a function of dose, drug concentration, and in the presence and absence of oxygen. It is more effective in protecting against sugar-phosphate damage under hypoxic conditions. The chemical modification is greater than that of cell killing as measured by the loss of colony-forming ability. Dose reduction factors greater than two are observed for DNA radioprotection, but the values in cells are much lower. These findings will be discussed in terms of the molecular mechanisms and their implications.

Direct photoaffinity labeling of an allosteric site of subunit protein M1 of mouse ribonucleotide reductase by dATP. Evidence for two independent binding interactions within the allosteric specificity site.

The M1 subunit of ribonucleotide reductase contains two kinds of allosteric sites, the activity site and the specificity site, which regulate the overall catalytic activity and the substrate specificity of the enzyme, respectively. The effector nucleotides, dGTP and dTTP, bind only to the specificity site; dATP and ATP bind to both sites. Partially purified protein M1 was photolabeled specifically after UV irradiation in the presence of [32P]dATP. The labeling occurred exclusively at the allosteric specificity site as evidenced by 1) total inhibition of the labeling by dGTP and dTTP, 2) normal photoincorporation of [32P]dATP by mutant protein M1 molecules that lack a functional activity site, and 3) coidentity of one-dimensional peptide maps of protein M1 labeled with either [32P]dATP or [32P]dTTP. A mutant protein M1 that is resistant to normal regulation by dGTP and dTTP (indicating an alteration in the allosteric specificity site) showed normal photoincorporation of [32P]dATP (but not [32P]dTTP). This labeling was not inhibited by dGTP or dTTP. Our data suggest that this mutation has altered the binding of dGTP and dTTP but not dATP (or ATP) at the specificity site. Thus, by the combination of genetic and photolabeling techniques, two independent nucleotide binding interactions occurring within this one complex regulatory domain can be distinguished.