Characterization of a cross-linked DNA-endonuclease VIII repair complex by electrospray ionization mass spectrometry.

Electrospray mass spectrometry techniques were used to characterize components of the active site in Endonuclease VIII by identifying the amino acid sequence and the binding site for a tryptic peptide derived from Endo VIII in a cross-linked DNA-peptide complex. Endo VIII, a DNA repair enzyme with both glycosylase and lyase activities, was covalently bound to a thymidine glycol-containing oligodeoxynucleotide duplex by converting a transient Schiff base formed during the course of the glycosylase activity to a stable covalent bond by chemical reduction with sodium borohydride. After tryptic digestion of the initial product, the identification of the cross-linked peptide was deduced initially from the molecular mass of the tryptic product obtained by negative ion electrospray mass analysis. Nanospray tandem mass spectrometry (MS/MS) analysis of the tryptic product corroborated the molecular mass of the peptide fragment and verified the point of attachment to the oligomer, but failed to produce sufficient fragmentation to sequence the peptide completely. Direct evidence for the amino acid sequence of the peptide was obtained after enzymatic digestion of the DNA portion of the cross-linked DNA-peptide product and analysis by negative ion nanospray MS/MS. Examination of the ions from collision induced fragmentation disclosed that this substance was the N-terminal tryptic fragment of Endo VIII cross-linked to a portion of the oligomer, and that the N-terminal proline from Endo VIII was covalently bound to the residual deoxyribose moiety at the original location of the thymine glycol in the oligomer.

3,N(4)-ethano-2'-deoxycytidine: chemistry of incorporation into oligomeric DNA and reassessment of miscoding potential.

3,N(4)-Ethano-2'-deoxycytidine (ethano-dC) may be incorporated successfully into synthetic oligodeoxynucleotides by omitting the capping procedure used in the automated DNA synthetic protocols immediately after inserting the lesion and in all iterations thereafter. Ethano-dC is sensitive to acetic anhydride found in the capping reagent, and multiple oligomeric products are formed. These products were identified by examining the reaction of ethano-dC with the capping reagent, and several acetylated, ring-opened products were characterized by electrospray mass spectrometry and collision induced dissociation experiments on a tandem quadrupole mass spectrometer. A scheme for the formation of the acetylated products is proposed. In addition, the mutagenic profile of ethano-dC was re-examined and compared to that for etheno-dC. Ethano-dC is principally a blocking lesion; however, when encountered by the exo(-)Klenow fragment of DNA polymerase, dAMP (22%), TMP (16%), dGMP (5.3%) and dCMP (1.2%) were all incorporated opposite ethano-dC, along with an oligomer containing a one-base deletion (0.6%).

8-Amino-2'-deoxyguanosine incorporation into oligomeric DNA.

During the incorporation of 8-amino-dG into oligomeric DNA, the deprotection conditions previously recommended (28% ammonia at room temperature) do not effect complete removal of the dimethylaminomethylene protecting groups. At elevated temperatures oxidative degradation of the oligomer and exchange of ammonia with dimethylamine in the protecting group at C8 occurred. The resolution of these problems and a method to obtain a series of homogeneous oligomers in reasonable yield containing 8-amino-dG located site-specifically are described.

Synthesis of enzymatically noncleavable carbocyclic nucleosides for DNA-N-glycosylase studies.

Carbocyclic nucleosides have been of great interest as antiviral agents and in studies in the area of antisense technology. The recent finding that the replacement of a single 2'-deoxynucleoside in DNA by a carba analogue does not alter the Watson-Crick base pairing, yet at the same time provides a chemically and enzymatically stable "glycosidic" linkage, led us to examine this class of compound as enzyme inhibitors of the DNA-repair enzymes involved in oxidative damage. We now report the synthesis and incorporation into oligomeric DNA via suitable derivatives, the carbanucleosides 8-oxo-7,8-dihydro-2'-deoxycarbainosine, 8-oxo-7,8-dihydro-2'-deoxycarbaguanosine, and 2'-deoxyaristeromycin. Aristeromycin (1) was deoxygenated at the 2'-position as follows. Treatment of 1 with TPDSCl2 gave the 3',5'-protected derivative 3 (76%) which on phenylthiocarbonylation at the 2'-position gave 4 in 51% yield. The latter compound on reduction with Bu3SnH led to the 2'-deoxy derivative 5 (90%). Benzoylation followed by deprotection with TBAF in THF then gave the desired intermediate (6) in 65% yield. N2-Isobutyryl-8-oxo-7,8-dihydro-2'-deoxycarbaguanosine (16) was synthesized from 3-chloro-2'-deoxycarbainosine (9). Treatment of 9, either with hydrazine followed by catalytic reduction of the 2-hydrazino derivative or with 1-(2-nitrophenyl)ethylamine followed by photolysis of the resulting 2-substituted derivative, in both instances gave the desired 2'-deoxycarbaguanosine (12) in approximately 50% overall yield in each case. Bromination of 12 gave 13 (90%) which, when treated with BnONa in DMSO at 65 degrees C, led to the 8-benzyloxy derivative 14 (46%). Isobutyrylation of 14 followed by catalytic reduction then afforded 16. 8-Oxo-7,8-dihydro-2'-deoxycarbainosine (23) was prepared in four steps. Bromination of 2'-deoxyaristeromycin (19) at the 8-position gave 20 (> 95%) which was converted to the 8-benzyloxy derivative 21 (61%) using BnONa/DMSO at 80 degrees C. Reductive debenzylation of 21 then led to 8-oxo-7,8-dihydro-2'-deoxyaristeromycin (approximately 100%) which, when treated with adenosine deaminase, provided the desired carbainosine derivative 23 in quantitative yield. Compounds 6, 16, and 23 were converted to their respective 5'-O-DMT, 3'-O-[(2-cyanoethoxy)-(N,N-diisopropylamino)phosphine] derivatives (8, 18, and 25) in excellent overall yields. The latter were then used to synthesize a series of DNA oligomers by automated procedures.

NH2-terminal proline acts as a nucleophile in the glycosylase/AP-lyase reaction catalyzed by Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) protein.

Formamidopyrimidine-DNA glycosylase (Fpg) protein plays a prominent role in the repair of oxidatively damaged DNA in Escherichia coli. The protein possesses three enzymatic activities, hydrolysis of the N-glycosidic bond (DNA glycosylase), beta-elimination (AP lyase), and delta-elimination; these functions act in a concerted manner to excise oxidized deoxynucleosides from duplex DNA. Schiff base formation between the enzyme and substrate has been demonstrated (Tchou, J., and Grollman, A. P. (1995) J. Biol. Chem. 270, 11671-11677); this protein-DNA complex can be trapped by reduction with sodium borohydride. By digesting the stable, covalently linked intermediate with proteases and determining the accurate mass of the products by negative electrospray ionization-mass spectrometry, we show that the N-terminal proline of Fpg protein is linked to DNA and, therefore, is identified as the nucleophile that initiates the catalytic excision of oxidized bases from DNA. This experimental approach may be applicable to the analysis of other protein-DNA complexes.

Characterization of the alkaline degradation products of an oligodeoxynucleotide containing 8-oxo-7,8-dihydro-2'-deoxyguanosine by electrospray ionization mass spectrometry.

Oligodeoxynucleotides containing 8-oxo-7,8-dihydro-2'-deoxyguanosine exhibit alkaline sensitivity and undergo cleavage of the phosphodiester backbone. Identification of the major degradation products and unstable intermediates formed in concentrated ammonia was accomplished by HPLC isolation and characterization by electrospray ionization mass spectrometry. Unstable intermediates were reduced in situ with NaBH4 prior to isolation and mass analysis. This technique produced accurate mass data for an oligonucleotide intermediate containing an abasic site, a strand cleavage, product containing the 3'-terminus, and two products with the 5'-terminus. 8-Oxoguanine was not present in the product HPLC chromatogram, suggesting rearrangement or degradation of this moiety prior to glycosidic bond cleavage. A scheme for the decomposition of 7,8-dihydro-2'-deoxyguanosine-containing oligonucleotides in 28% ammonia solution is presented.

Analysis of synthetic oligodeoxynucleotides containing modified components by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI/MS) has been used to confirm the synthesis of oligodeoxynucleotides containing modified structures and to identify products of unstable oligomer degradation. Negative ion mass spectra of oligomers and dimethoxytrityl-protected oligomers exhibited a distribution of multiply charged molecular ions with few adduct ions present. Molecular masses may be determined from the array of peaks with excellent accuracy and serve to corroborate the synthetic sequence and the inclusion of a modified structure within the sequence. Incorporation of modified deoxynucleotides, stable isotopes, and other novel structures such as a terminal biotin moiety and a phosphorothioate linkage can be rapidly verified by ESI/MS. This technique has also proved useful for the identification of products of unstable oligomers formed during synthesis, deprotection, or purification, including the major products formed under basic conditions from 5'-dimethoxytrityl-protected oligomers containing 8-oxo-2'-deoxyguanosine.

Characterization of thymidine adducts formed by acrolein and 2-bromoacrolein.

Thymidine was permitted to react with the known mutagens acrolein and 2-bromoacrolein under physiological conditions. The products of these reactions were separated by HPLC and characterized by UV, FAB/MS, electrospray MS, 1H NMR and chemical transformation. The reaction with acrolein gave one major product, N3-(3''-oxopropyl)thymidine, which is unstable in aqueous solution and was reduced with sodium borohydride to the corresponding alcohol. Reaction with 2-bromoacrolein yielded the unstable intermediate, N3-(2''-bromo-3''-oxopropyl)thymidine, and two stable products, the diastereomers of N3-(2''-hydroxy-3''-oxopropyl)thymidine, which are slowly transformed to N3-(2''-oxo-3''-hydroxypropyl)thymidine. Reactions with both mutagens proceed most rapidly at pH 9.2, less rapidly at pH 7.4, and no products are found at pH 4.2. Stable adducts found in the reaction of 2-bromoacrolein were also identified in reactions with single-strand oligodeoxynucleotides using a sensitive, selected ion monitoring GC/MS procedure.

Strength of thin chemtempered lenses: static load testing.

Static load tests were conducted on heat-tempered and chemtempered plano white crown glass lenses from five different optical laboratories. With both ball-on-ring and ring-on-ring loading, chemtempered lenses considerably thinner than 2.0 mm were found to be as failure resistant as 2.0-mm-thick heat-tempered lenses. A similar result was obtained previously using the drop-ball test. It is shown that the theory of brittle fracture can be used to relate the results of different tests and provides a rational basis for comparing the relative performance of chemtempered and heat-tempered lenses.

Strength of thin chemtempered lenses: drop-ball testing.

Failure heights were measured in drop-ball tests for both chemtempered and heat-tempered plano, white crown glass lenses from five different optical laboratories. It was found that (1) failure height was proportional to the square of the lens thickness, (2) chemtempered lenses substantially thinner than 2.0 mm are as resistant to breakage as 2.0-mm-thick heat-tempered lenses, and (3) a close correlation existed between results of single-drop and multiple-drop tests and between results of tests using rigid and compliant mounts.

Basic principles of lens fracture testing.

An analysis of different tests used or proposed for evaluation of the resistance of ophthalmic lenses to breakage is presented. The tests considered subject the lenses to different strain rates, areas under stress, and energy losses. The analysis examines effects of these variables as well as effects of differences in lens-failure stress and lens thickness on test results.