The effects of covalent additions of a psoralen on transcription by<i>E. coli</i>RNA polymerase

Shi, Yun‐Bo; Gamper, Howard; Hearst, John E.

doi:10.1093/nar/15.17.6843

Cited by 51 publications

(39 citation statements)

References 45 publications

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“…Formation ofpsoralen photoproducts (15)(16)(17), as well as a stable triplex on a duplex target, has been shown to inhibit transcription (18,19). On a single-stranded nucleic acid, triplex formation might be expected to be more efficient in arresting biological processes than formation of a double helix with an antisense oligonucleotide.…”

Section: Resultsmentioning

confidence: 99%

Oligonucleotide clamps arrest DNA synthesis on a single-stranded DNA target.

Giovannangéli

Thuong

Hélène

1993

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

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Triple helices can be formed on singlestranded oligopurine target sequences by composite oligonucleotides consisting of two oligonucleotides covalently linked by either a hexaethylene glycol linker or an oligonucleotide sequence. The first oligomer forms Watson-Crick base pairs with the target, while the second oligomer engages in Hoogsteen base pairing, thereby acting as a molecular clamp. The triple-helical complex formed by such an oligonucleotide clamp, or "oligonucleotide4oop-oligonucleotide" (OLO), is more stable than either the corresponding trimolecular triple helix or the double helix formed upon binding of the oligopyrimidine complement to the same oligopurine target. Attaching a psoralen derivative to the 5' end of the OLO allowed us to photoinduce a covalent linkage to the target sequence. The psoralen moiety became covalently linked to all three portions of the triplex, thereby making the oligonucleotide clamp irreversible. These crosslinking reactions introduced strong stop signals during DNA replication, as shown on a plasmid containing a portion of the HIV proviral sequence of human immunodeficiency virus. A 16-mer oligopurine sequence corresponding to the "polypurine tract" of human immunodeficiency virus was chosen as a target for a psoralen-OLO conjugate. Three

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Section: Resultsmentioning

confidence: 99%

Oligonucleotide clamps arrest DNA synthesis on a single-stranded DNA target.

Giovannangéli

Thuong

Hélène

1993

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

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“…Human excision nuclease releases excised oligomers from gapped duplex. The 3Ј-terminally biotinylated, internally radiolabeled cholesterol-A substrate 156 nucleotides in length Shi et al, 1987) was incubated with E. coli or human excision nuclease. The DNA was then pulled down with streptavidin beads, and the streptavidin bead-bound (B) and -unbound (U) fractions were analyzed on a sequencing gel.…”

Section: Discussionmentioning

confidence: 99%

Reaction Mechanism of Human DNA Repair Excision Nuclease

Mu¹,

Hsu²,

Sancar

1996

Journal of Biological Chemistry

351

329

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Nucleotide excision repair consists of removal of the damaged nucleotide(s) from DNA by dual incision of the damaged strand on both sides of the lesion, followed by filling of the resulting gap and ligation. In humans, 14 -16 polypeptides are required for the dual incision step. We have purified the required proteins to homogeneity and reconstituted the dual incision activity (excision nuclease) in a defined enzyme/substrate system. The system was highly efficient, removing >30% of the thymine dimers under optimal conditions. All of the six fractions that constitute the excision nuclease were required for dual incision of the thymine dimer substrate. However, when a cholesterol-substituted oligonucleotide was used as substrate, excision occurred in the absence of the XPC-HHR23B complex, reminiscent of transcription-coupled repair in the XP-C mutant cell line. Replication protein A is absolutely required for both incisions. The XPG subunit is essential to the formation of the preincision complex, but the repair complex can assemble and produce normal levels of 3-incision in the absence of XPF-ERCC1. Kinetic experiments revealed that the 3-incision precedes the 5-incision. Consistent with the kinetic data, uncoupled 5-incision was never observed in the reconstituted system. Two forms of TFIIH were used in the reconstitution reaction, one containing the CDK7-cyclin H pair and one lacking it. Both forms were equally active in excision. The excised oligomer dissociated from the gapped DNA in a nucleoprotein complex. In total, these results provide a detailed account of the reactions occurring during damage removal by human excision nuclease.

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“…Toward this goal, Selby and Sancar (37) adapted the transcription-repair template/substrate system of Shi et al (42) to the study of thymine dimer (T< >T) repair in a defined system. It was found that a T< >T in the coding (nontranscribed) strand had no discernible effect on transcription.…”

Section: Introductionmentioning

confidence: 99%