Preparation and Characterization of a Deoxyoligonucleotide 49-mer Containing a Site-Specific Thymidylyl-3',5'-adenosine Photoproduct

Zhao, Xiaodong; Kao, Jeffrey L.-F.; Taylor, John‐Stephen

doi:10.1021/bi00004a033

Cited by 24 publications

(21 citation statements)

References 19 publications

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“…By incorporating a single TA* photolesion into a 49-mer oligonucleotide (28) and thence into an M13mp18-derived phage construct, Zhao and Taylor (33) were able to assess the mutagenicity of TA* in transfected Escherichia coli cells. In the absence of the SOS response, the main effect of TA* was to block DNA replication.…”

Section: Discussionmentioning

confidence: 99%

High-resolution crystal structure of the intramolecular d(TpA) thymine-adenine photoadduct and its mechanistic implications

Davies

Malone

Gan

et al. 2007

Nucleic Acids Research

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A high-resolution crystal structure is reported for d(TpA)*, the intramolecular thymine–adenine photoadduct that is produced by direct ultraviolet excitation of the dinucleoside monophosphate d(TpA). It confirms the presence of a central 1,3-diazacyclooctatriene ring linking the remnants of the T and A bases, as previously deduced from heteronuclear NMR measurements by Zhao et al. (The structure of d(TpA)*, the major photoproduct of thymidylyl-(3′-5′)-deoxyadenosine. Nucleic Acids Res., 1996, 24, 1554–1560). Within the crystal, the d(TpA)* molecules exist as zwitterions with a protonated amidine fragment of the eight-membered ring neutralizing the charge of the internucleotide phosphate monoanion. The absolute configuration at the original thymine C5 and C6 atoms is determined as 5S,6R. This is consistent with d(TpA)* arising by valence isomerization of a precursor cyclobutane photoproduct with cis–syn stereochemistry that is generated by [2 + 2] photoaddition of the thymine 5,6-double bond across the C6 and C5 positions of adenine. This mode of photoaddition should be favoured by the stacked conformation of adjacent T and A bases in B-form DNA. It is probable that the primary photoreaction is mechanistically analogous to pyrimidine dimerization despite having a much lower quantum yield.

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Section: Discussionmentioning

confidence: 99%

High-resolution crystal structure of the intramolecular d(TpA) thymine-adenine photoadduct and its mechanistic implications

Davies

Malone

Gan

et al. 2007

Nucleic Acids Research

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“…The exo Ϫ T7 polymerases have been reported to bypass the bulky 2-aminofluorene (AF) adduct of guanine (35,36), and the bulky 7-bromomethylbena[a]anthracene (37) and styrene oxide (38) adducts of adduct of dA. The exo Ϫ -deficient T7 polymerase also bypasses photochemically cross-linked TA sites (39), and cis-diamminedichloroplatinum(II) cross-linked purines in GG, AG, and GCG (40) though no bypass was observed in different sequence context for the GG and AG sites (41). On the other hand, exo Ϫ T7 polymerase has not been found to bypass acetylaminofluorene (AAF) (32,36) or benzopyrenediolexpoxide (BPDE) (42) adducts.…”

Section: Discussionmentioning

confidence: 99%

The Ability of a Variety of Polymerases to Synthesize Past Site-specific cis-syn, trans-syn-II, (6–4), and Dewar Photoproducts of Thymidylyl-(3′→5′)-thymidine

Smith

Baeten

Taylor

1998

Journal of Biological Chemistry

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The role of photoproduct structure, 3 3 5 exonuclease activity, and processivity on polynucleotide synthesis past photoproducts of thymidylyl-(3 3 5)-thymidine was investigated. Both Moloney murine leukemia virus reverse transcriptase and 3 3 5 exonuclease-deficient (exo ؊ ) Vent polymerase were blocked by all photoproducts, whereas Taq polymerase could slowly bypass the cis-syn dimer. T7 RNA polymerase was able to bypass all the photoproducts in the order cis-syn > Dewar > (6 -4) > trans-syn-II. Klenow fragment could not bypass any of the photoproducts, but an exo ؊ mutant could bypass the cis-syn dimer to a greater extent than the others. Likewise T7 DNA polymerase, composed of the T7 gene 5 protein and Escherichia coli thioredoxin, was blocked by all the photoproducts, but the exo ؊ mutant Sequenase 2.0 was able to bypass them all in the order cissyn > Dewar > trans-syn-II > (6 -4). No bypass occurred with an exo ؊ gene 5 protein in the absence of the thioredoxin processivity factor. Bypass of the cis-syn and trans-syn-II products by Sequenase 2.0 was essentially non-mutagenic, whereas about 20% dTMP was inserted opposite the 5-T of the Dewar photoproduct. A mechanism involving a transient abasic site is proposed to account for the preferential incorporation of dAMP opposite the 3-T of the photoproducts.Dipyrimidine sites are the major sites of UV-induced photoproducts and mutations (1-7). The four main classes of photoproducts formed by ultraviolet light at dipyrimidine sites (shown in Fig. 1 for a TpT 1 site) are the cis-syn and trans-syn (trans-syn-I (8) and trans-syn-II (9)) cyclobutane dimers and the (6 -4) pyrimidine-pyrimidone photoproducts and their Dewar valence isomers (10 -13). All of these photoproducts have been found to lead to mutations in Escherichia coli under SOS conditions by use of site-specific photoproduct-containing bacteriophage vectors, but the (6 -4) and Dewar photoproducts are far more mutagenic than either the cis-syn and trans-syn isomers (14 -18). The extent to which a particular photoproduct contributes to UV-induced mutations at a particular site not only depends on its mutagenicity, but also depends on its rate of induction, repair, and DNA synthesis bypass (7,12). At the moment, the relative contribution of individual DNA photoproducts to UV-induced mutations is not known, nor are the detailed mechanisms by which DNA photoproducts are repaired or bypassed. Recently, we have prepared homogeneous 49-mer oligonucleotides containing the four major photoproduct classes of TpT (19) 2 for use as substrates for the necessary in vitro and in vivo mechanistic studies. Herein, we report the use of these 49-mers and 72-mers containing a T7 promoter to study the role of photoproduct structure and the 3Ј 3 5Ј exonuclease activity and processivity of polymerases on DNA and RNA synthesis past these photoproducts. EXPERIMENTAL PROCEDURESEnzymes, Reagents, and Equipment-The preparation of the photoproduct-containing 49-mers has been reported elsewhere (19).2 Other oligonucleotides were purchas...

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“…1 B and C). Oligonucleotides were obtained from Operon Technologies (Alameda, CA) or were prepared in house (36,37).…”

Section: Methodsmentioning

confidence: 99%

Repair of DNA–polypeptide crosslinks by human excision nuclease

Reardon

Sancar

2006

Proc. Natl. Acad. Sci. U.S.A.

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DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4-and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome.damage recognition ͉ nucleotide excision repair D NA-protein crosslinks are intermediates in the reaction pathways of certain enzymes such as integrases, topoisomerases, and meiotic recombinases (1-4). In addition, crosslinks are induced by DNA-damaging agents including ionizing radiation, UV light, metals such as chromium and arsenic (5, 6), and bifunctional chemotherapeutic drugs such as platinum compounds and nitrogen mustards (7-9). Finally, crosslinks may form when repair enzymes attempt to process aldehyde groups either in the DNA backbone in the form of apurinic͞ apyrimidinic (AP) sites or on oxidized nucleobase residues (10).The repair of some of these DNA-protein crosslinks is well understood: the topoisomerase I-DNA 3Ј tyrosyl-phosphodiester is commonly cleaved in the normal course of the topoisomerase (topo) reaction. However, when topo cleaves DNA near lesions such as pyrimidine dimers or nicks, the enzyme-DNA crosslink is frozen in transit, giving rise to a stable crosslink at a single-stranded nick site. This lesion is repaired by a remarkable enzyme called tyrosyl-DNA phosphodiesterase, Tdp1 (1). Tdp1 cleaves the 3Ј tyrosyl-phosphodiester bond and converts it to a 3Ј phosphate terminus, which is then processed to a 3Ј OH that can be ligated. Tdp1 has also been implicated in the removal of other 3Ј adducts (2). The enzyme has been found in all eukaryotes tested in which a topo I-3Ј tyrosylphosphodiester bond forms in the course of the reaction but not in prokaryotes in which the reaction intermediate is a topo I-DNA 5Ј tyrosyl-phosphodiester (3).DNA-protein crosslinks can also form during meiosis. Spo11 in yeast (SPO11 in mammals) makes a staggered double-strand break to initiate meiotic recombination; each subunit of the dimeric protein is linked to the resulting 5Ј ends through a 5Ј tyrosyl-phosphodiester linkage. In contrast to the topo I-3Ј tyrosyl-phosphodiester complex, the Spo11-...

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Preparation and Characterization of a Deoxyoligonucleotide 49-mer Containing a Site-Specific Thymidylyl-3',5'-adenosine Photoproduct

Cited by 24 publications

References 19 publications

High-resolution crystal structure of the intramolecular d(TpA) thymine-adenine photoadduct and its mechanistic implications

High-resolution crystal structure of the intramolecular d(TpA) thymine-adenine photoadduct and its mechanistic implications

The Ability of a Variety of Polymerases to Synthesize Past Site-specific cis-syn, trans-syn-II, (6–4), and Dewar Photoproducts of Thymidylyl-(3′→5′)-thymidine

Repair of DNA–polypeptide crosslinks by human excision nuclease

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