Synthesis of a<i>trans-syn</i>thymine dimer building block. Solid phase synthesis of CGTAT[<i>t,s</i>]TATGC

Taylor, John‐Stephen; Brockie, Ian R.

doi:10.1093/nar/16.11.5123

Cited by 52 publications

(46 citation statements)

References 12 publications

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“…The primer (pGCTCGAGCTCAAT<>TAGTCAG) was synthesized by the building-block method (19,20 (21). Double-stranded DNA was purified from products of incomplete synthesis (22).…”

Section: Methodsmentioning

confidence: 99%

Differential replication of a single, UV-induced lesion in the leading or lagging strand by a human cell extract: fork uncoupling or gap formation.

Svoboda

Vos

1995

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

104

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We have constructed simian virus 40 minireplicons containing uniquely placed cis,syn-thymine dimers (T< >T) for the analysis of leading-and lagging-strand bypass replication. Assaying for replication in a human cellfree extract through the analysis of full-size labeled product molecules and restriction fragments spanning the T< >T site resulted in the following findings: (i) Despite these investigations, a basic understanding of replication of damaged DNA in normal human cells is lacking (2).In vitro studies using primed single-stranded DNA templates and purified DNA polymerases (9-13) have demonstrated the existence of both stalled replication intermediates and translesion synthesis. These studies involved DNA synthesis on primed templates rather than semiconservative coordinated replication of leading and lagging strands at a bona fideThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. replication fork. In vitro replication of SV40-based minireplicons carrying randomly distributed, UVC-induced Pyr<>Pyr has been analyzed in human cell extracts (14,15). A UV fluence-dependent inhibition of covalently closed circular DNA (form I DNA) synthesis was observed, which confirmed the blocking effect of such lesions on DNA replication. The presence of a UV endonuclease V from phage T4 (T4 UV endo)-sensitive product implied that replication of Pyr<>Pyr occurred in such a system. A detailed examination of the molecular mechanism of UV damage processing has become possible with the development of site-specific lesioncontaining DNA (11-13). Thus, we now report sites of blockage and rates of bypass of individual lesions in the leading and lagging strands.The most prevalent DNA modification resulting from UVC irradiation is the cis,syn-thymine dimer (T<>T), which has been a paradigm DNA lesion (16). We have prepared plasmids containing the SV40 origin of replication together with uniquely placed T<>T and have analyzed their in vitro replication in a human cell-free extract. We have developed these assays to study the mechanism of replication of DNA damaged in either the leading or lagging strands of a human replication fork. Specifically, we have determined the site of blockage of strand elongation on T<>T-containing DNA templates, compared the bypass of T<>T in the leading and lagging strands, and analyzed the potential uncoupling of coordinated leading-and lagging-strand synthesis at T<>T-blocked replication forks. MATERIALS AND METHODS

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“…The primer (pGCTCGAGCTCAAT<>TAGTCAG) was synthesized by the building-block method (19,20 (21). Double-stranded DNA was purified from products of incomplete synthesis (22).…”

Section: Methodsmentioning

confidence: 99%

Differential replication of a single, UV-induced lesion in the leading or lagging strand by a human cell extract: fork uncoupling or gap formation.

Svoboda

Vos

1995

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

104

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“…Therefore, we have reexamined this question, employing new methodology that has become available within the last few years-specifically, the ability to make large quantities of substrate with a single TOT at a defined position (13 (14).…”

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confidence: 99%

“…Two types of substrates were used: a synthetic oligomer with a single TOT, and UV-irradiated pBR322. The synthetic substrate was prepared by the "building block" method (13) and was a gift of J.-S. Taylor and D. L. Svoboda (Washington University, St. Louis). The oligomer containing TOT was 20 nucleotides in length: 5'-GCTCGAGCTATOTAACGTCAG-3'.…”

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confidence: 99%

Evidence for lack of DNA photoreactivating enzyme in humans.

Sancar

1993

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

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Photoreactivating enzyme (DNA photolyase; deoxyribocyclobutadipyrimidine pyrimidine-lyase, EC 4.1.99.3) repairs UV damage to DNA by utilizing the energy of near-UV/visible light to split pyrimidine dimers into monomers. The enzyme is widespread in nature but is absent in certain species in a seemingly unpredictable manner. Its presence in humans has been a source of considerable controversy. (2). Similarly, primary fibroblasts from the rattlesnake (Crotalus horridus) remove thymine dimers (TOT) by photoreactivation efficiently, whereas those from the garter snake (Thamnophis sp.) do not (3).When mammalian cells were tested, initially it was claimed that marsupials were capable ofphotoreversing YOY but that placental animals, including humans, were not (4, 5). However, subsequent research on this subject yielded conflicting results. In a series of papers, it was reported that human leukocytes as well as human and murine cells in culture contained photolyase (6-8), that fibroblasts from a patient of xeroderma pigmentosum (XP) complementation group B were completely defective in the enzyme (7), and that cell lines from other XP complementation groups, including the XP variant, had diminished photoreactivation activity (7,8).Other studies failed to detect photoreactivating enzyme (DNA photolyase) in human or murine cell lines (9). However, evidence was presented suggesting that expression of the enzyme is tissue-specific and that in tissue culture the expression is influenced by the growth medium (10, 11), which may account for the failure of some researchers to detect photoreactivation in humans and other placental mammals.The presence or absence of photolyase in humans is important from the standpoint of public health (12 (14).Photolyase Substrates. Two types of substrates were used: a synthetic oligomer with a single TOT, and UV-irradiated pBR322. The synthetic substrate was prepared by the "building block" method (13) and was a gift of J.-S. Taylor and D. L. Svoboda (Washington University, St. Louis). The oligomer containing TOT was 20 nucleotides in length: 5'-GCTCGAGCTATOTAACGTCAG-3'. To prepare substrate, this 20-mer was 5'-end-labeled, annealed to the complementary strand, and digested with Mse I (which incises at TITAA) to eliminate dimer-free contaminant. The resulting DNA was >99% pure with regard to TOT content. The concentration of the substrate used in Figs. 2 and 3 was determined by Cerenkov counting and by taking into account that 50%o of the label used in the kinase reaction was incorporated into DNA. The specific activity of this substrate was 6835 Ci/mmol.The pBR322 substrate was prepared by irradiating the plasmid with 254-nm light and the number of YOY per plasmid molecule was determined by a transformation assay (14).

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“…Custom made DNA oligonucleotides were purchased from Qiagen. Oligonucleotides containing site-specific CPDs, constructed as described previously (20,21), were obtained from Dr. JohnStephen Taylor (Washington University, St. Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

Transcription Arrest at a Lesion in the Transcribed DNA Strand in Vitro Is Not Affected by a Nearby Lesion in the Opposite Strand

Kalogeraki

Tornaletti

Hanawalt

2003

Journal of Biological Chemistry

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Cis-syn cyclobutane pyrimidine dimers (CPDs) are the most frequently formed lesions in UV-irradiated DNA. CPDs are repaired by the nucleotide excision repair pathway. Additionally, they are subject to transcriptioncoupled DNA repair. In the general model for transcription-coupled DNA repair, an RNA polymerase arrested at a lesion on the transcribed DNA strand facilitates repair by recruiting the repair machinery to the site of the lesion. Consistent with this model, transcription experiments in vitro have shown that CPDs in the transcribed DNA strand interfere with the translocation of prokaryotic and eukaryotic RNA polymerases. Here, we study the behavior of RNA polymerase when transcribing a template that contains two closely spaced lesions, one on each DNA strand. Similar DNA templates containing no CPD, or a single CPD on either the transcribed or the nontranscribed strand were used as controls. Using an in vitro transcription system with purified T7 RNA polymerase (T7 RNAP) or rat liver RNAP II, we characterized transcript length and efficiency of transcription in vitro. We also tested the sensitivity of the arrested RNAP II-DNA-RNA ternary complex, at a CPD in the transcribed strand, to transcription factor TFIIS. The presence of a nearby CPD in the nontranscribed strand did not affect the behavior of either RNA polymerase nor did it affect the reverse translocation ability of the RNAP II-arrested complex. Our results additionally indicate that the sequence context of a CPD affects the efficiency of T7 RNAP arrest more significantly than that of RNAP II.It has been nearly two decades since the discovery that in UV-irradiated cells of eukaryotes and prokaryotes the transcribed strand of an active gene is repaired more rapidly than the nontranscribed strand (1, 2). However, the mechanistic details of this phenomenon, termed transcription-coupled repair (TCR), 1 are still not fully understood. The general model for the mechanism of TCR postulates that an RNA polymerase stalled at a lesion is the signal for recruitment of the repair proteins and initiation of excision repair (3). In the case of UV-induced DNA lesions, such as the most frequently formed cis-syn CPDs, the specific repair process requires that an oligonucleotide containing the damaged site is excised from the DNA, the gap created is closed by repair replication, and finally the repair patch is ligated to the contiguous DNA (reviewed in Refs. 4 and 5). An active RNA polymerase is a prerequisite for TCR (6 -8). However, the arrested polymerase must then be displaced so that the lesion becomes accessible to the repair machinery (3). In Escherichia coli, a transcription-repair coupling factor (Mfd) has been shown to displace the stalled polymerase and facilitate TCR (9). In eukaryotes, however, it is still not clear how the arrested polymerase signals the assembly of the repair machinery at the site of damage, nor is it clear how the polymerase is displaced to allow access of the repair proteins to the damaged site. Despite the evidence that incomple...

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Synthesis of atrans-synthymine dimer building block. Solid phase synthesis of CGTAT[t,s]TATGC

Cited by 52 publications

References 12 publications

Differential replication of a single, UV-induced lesion in the leading or lagging strand by a human cell extract: fork uncoupling or gap formation.

Differential replication of a single, UV-induced lesion in the leading or lagging strand by a human cell extract: fork uncoupling or gap formation.

Evidence for lack of DNA photoreactivating enzyme in humans.

Transcription Arrest at a Lesion in the Transcribed DNA Strand in Vitro Is Not Affected by a Nearby Lesion in the Opposite Strand

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