The repair of psoralen monoadducts by the<i>Escherichia coli</i>UvrABC endonuclease

Yeung, Anthony T.; Jones, Beverly K.; Capraro, Michael A.; Chu, Tim

doi:10.1093/nar/15.12.4957

Cited by 39 publications

(32 citation statements)

References 18 publications

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“…react most efficiently at thymidines in 5Ј-TpA sequences, followed by 5Ј-ApT and 5Ј-TpG sequences (9,54). Adducts have been reported at cytidines in 5Ј-CpA and 5Ј-ApC contexts, yet modification of 5Ј-CpG has not been observed (21).…”

Section: Discussionmentioning

confidence: 99%

Strand Specificity of Mutagenic Bypass Replication of DNA Containing Psoralen Monoadducts in a Human Cell Extract

Thomas

Svoboda

Vos

et al. 1996

Molecular and Cellular Biology

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Psoralens are mutagenic compounds of vegetable origin that are used as photosensitizing agents in the treatment of various skin diseases, blood cell cancer, and autoimmune disorders. To study the mechanism of mutagenicity of psoralens in humans, we examined the efficiency and fidelity of simian virus 40 origindependent replication in a human cell extract of M13mp2 DNA randomly treated with the psoralen derivative 4-hydroxymethyl-4,5,8-trimethyl psoralen plus UVA irradiation. Replication of DNA treated with variable amounts of 4-hydroxymethyl-4,5,8-trimethyl psoralen and a fixed UVA fluence was inhibited in a concentration-dependent manner. However, covalently closed monomer-length circular replication products were observed. Product analysis by renaturing agarose gel electrophoresis after cross-linking with 250-to 280-nm UV light indicated that approximately 1 of 9 psoralen monoadducts was bypassed during in vitro replication. Introduction of product DNA into Escherichia coli to score replication errors in the lacZ␣ reporter gene demonstrated that replication of the damaged DNA was more mutagenic than was replication of undamaged DNA. Sequence analysis of lacZ mutants revealed that damage-dependent replication errors were predominantly T ⅐ A3C ⅐ G transitions, transversions at C ⅐ G base pairs, and deletions of single A ⅐ T base pairs, the last occurring most frequently in homopolymeric runs. A comparison of error specificities with two substrates having the replication origin asymmetrically placed on opposite sides of the mutational target suggests that the lagging-strand replication apparatus is less accurate than the leading-strand replication apparatus for psoralen monoadduct-dependent deletion errors. A model is proposed based on the preferential loopout of the monoadducted base from the strand that templates retrograde discontinuous synthesis.DNA replication in eukaryotic cells is a complex process requiring a variety of proteins to synthesize the leading and lagging strands in an asymmetric yet coordinated manner (14). Because of this asymmetry, DNA adducts that escape repair may have different potentials for blocking replication or promoting replication infidelity, depending on which strand the adduct is located on. For example, in mammalian cells deficient in excision repair, mutations were predominantly found on one of the two strands of the hprt gene (6,19,20,50,51), leading to the suggestion that the fidelity of translesion bypass is more error prone on the leading strand (20, 51).To study the processes that determine the fidelity of DNA replication, we have been using the SV40 replication system as a model for human chromosomal replication (for reviews, see references 29 and 37). Using substrates containing the SV40 origin of replication, we (27,30,31,41) and others (11) have shown that the replication apparatus in mammalian cell extracts is quite accurate when replicating undamaged DNA, with an error rate ranging from less than or equal to 6.2 ϫ 10 Ϫ6 to 1 ϫ 10 Ϫ7 error per nucleotide. These observ...

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Section: Discussionmentioning

confidence: 99%

Strand Specificity of Mutagenic Bypass Replication of DNA Containing Psoralen Monoadducts in a Human Cell Extract

Thomas

Svoboda

Vos

et al. 1996

Molecular and Cellular Biology

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“…Purified UvrABC endonuclease (10) incision conditions were the same as in our previous studies with other forms of substrates (3,8,11).…”

Section: Methodsmentioning

confidence: 99%

Repair of 4,5',8-trimethylpsoralen monoadducts and cross-links by the Escherichia coli UvrABC endonuclease.

Jones

Yeung

1988

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

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Using an oligonucleotide model substrate, we observed two unusual mechanisms of UvrABC endonuclease in the repair of 4,5',8-trimethylpsoralen monoadducts and crosslinks. (i) UvrABC endonuclease usually incises a psoralen monoadduct only on the damaged strand. However, for one of the monoadducts we studied, incision on the complementary undamaged strand was also observed at a very low frequency, as though the adduct were on the thymine across from the damaged strand. Although the details of the erroneous incision are not yet known, such erroneous incision is potentially mutagenic. (ii) In cross-link repair, we observed that the UvrABC endonuclease incises the cross-linked DNA on either the furan side strand or the pyrone side strand. The incisions are not equally efficient. These data suggest that the structure of a psoralen cross-link, as seen by a repair enzyme, varies with the DNA sequence.Psoralens are a family of photoreactive furocoumarins, planar three-ringed heterocyclic hydrocarbon compounds (1), which have been used to treat skin ailments such as psoriasis (2). 4,5',8-Trimethylpsoralen (TMP) is specific for 5'-TpA sequences and photoreacts with thymines in a 5'-TpA sequence in a DNA duplex to form first a furan side monoadduct and then an interstrand cross-link (3, 4). Both the monoadducts and the cross-links of psoralens are known to be repaired by nucleotide excision repair systems such as the UvrABC endonuclease system of Escherichia coli (5, 6).With a psoralen monoadduct, UvrABC endonuclease incises at the eighth phosphodiester moiety 5' to the modified base and at the fifth phosphodiester moiety 3' to the modified base (3, 7). In vitro, at least, the incisions are not always bimodal and precise. Sometimes, only one of the two incisions is made (8). The reason for these single incisions is not known. The 5' incision at a psoralen monoadduct lesion can also be at the ninth instead of the eighth phosphodiester moiety (3). This finding suggests that the sequences around a lesion may exist in more than one conformation, some or all ofwhich can be acted upon by the UvrABC endonuclease (3).Depending on the orientation of a psoralen molecule in a given 5'-TpA site, two isomers of psoralen cross-links exhibiting different DNA helix stabilities are formed (4). Our present study will determine whether the UvrABC endonuclease would respond differently to the two cross-linked isomers. This paper also provides enzymatic evidence that the structure ofa psoralen cross-link varies with the sequence of the DNA. To begin to examine how errors in DNA repair might be important to mutagenesis, we tested and observed that the UvrABC endonuclease can err and incise the undamaged DNA strand across from a psoralen monoadduct. METHODSPreparation of Homogeneous DNA Substrates Containing TMP Monoadducts and Cross-Links. To avoid UV damage, all oligonucleotides and substrates were purified from polyacrylamide gels by isolation of the bands located by autoradiography. All purification gels were run with the lightly label...

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“…The E. coli UvrABC endonuclease has been highly purified and characterized biochemically (1)(2)(3)(4)(5)(6)(7). In the presence of MgATP, UvrA and UvrB proteins form a high-affinity complex specifically with damaged DNA (2,7). UvrC protein binds to the UvrAB-DNA complex to form the UvrABC endonuclease that incises the altered DNA strand (2).…”

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“…It forms a high-affinity reversible complex with DNA. In E. coli, ditercalinium was cytotoxic only to a polA strain and to a lig 7 strain at nonpermissive temperature (12). The suppression of ditercalinium cytotoxicity in polA strains by the uvrA mutation suggested the involvement of the nucleotide excision repair process in the mechanism of cytotoxicity (12).…”

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confidence: 99%

The noncovalent complex between DNA and the bifunctional intercalator ditercalinium is a substrate for the UvrABC endonuclease of Escherichia coli.

Lambert

Jones²,

Roques³

et al. 1989

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

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We have demonstrated that the noncovalent complex formed between DNA and an antitumor bifunctional intercalator, ditercalinium, is recognized in vitro as bulky covalent DNA lesions by the purified Escherichia coli UvrABC endonuclease. It was established that no covalent drug-DNA adduct was formed during the incubation ofthe drug with DNA or during subsequent incubation with the UvrAB proteins. The nucleoprotein-ditercalinium complexes appear different from those generated by repair of pyrimidine dimers. The UvrA protein is able to form a stable complex with ditercaliniumintercalated DNA in the presence of ATP, whereas both UvrA and UvrB proteins are required to form a stable complex with pyrimidine dimer-containing DNA. The apparent half-life of the UvrA-and UvrAB-ditercalinium-DNA complexes following removal of free ditercalinium is 5 min. However, if the free ditercalinium concentration is maintained to allow the intercalation of one molecule of ditercalinium per 3000 base pairs, the half-life of the UvrA-or UvrAB-ditercalinium-DNA complex is 50 min, comparable to that of the complex of UvrAB proteins formed with pyrimidine dimer-containing DNA.UvrABC endonuclease incises ditercalinium-intercalated DNA as efficiently as pyrimidine dimer-containing DNA. However, unlike repair of pyrimidine diners, the incision reaction is strongly favored by the supercoiling of the DNA substrate. Because UvrA-or UvrAB-ditercalinium-DNA complexes can be formed with relaxed DNA without leading to a subsequent incision reaction, these apparently dead-end nucleoprotein complexes may become lesions in themselves resulting in the cytotoxicity of ditercalinium. Our results show that binding of excision repair proteins to a noncovalent DNA-ligand complex may lead to cell toxicity.

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The repair of psoralen monoadducts by theEscherichia coliUvrABC endonuclease

Cited by 39 publications

References 18 publications

Strand Specificity of Mutagenic Bypass Replication of DNA Containing Psoralen Monoadducts in a Human Cell Extract

Strand Specificity of Mutagenic Bypass Replication of DNA Containing Psoralen Monoadducts in a Human Cell Extract

Repair of 4,5',8-trimethylpsoralen monoadducts and cross-links by the Escherichia coli UvrABC endonuclease.

The noncovalent complex between DNA and the bifunctional intercalator ditercalinium is a substrate for the UvrABC endonuclease of Escherichia coli.

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