Removal of psoralen monoadducts and crosslinks by human cell free extracts

Reardon, Joyce T.; Spielmann, Peter; Huang, Juch Chin; Sastry, Srinivas S.; Sancar, Aziz; Hearst, John E.

doi:10.1093/nar/19.17.4623

Cited by 43 publications

(26 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…RPA binds to triplexes with high affinity, with or without XPA, whereas XPA binds only in conjunction with RPA, but contributes to specificity by selectively diminishing nonspecific binding and͞or aggregation of RPA to undamaged DNA. Psoralen monoadducts and crosslinks are, at least in part, recognized and repaired by NER in human cells (39)(40)(41)(42)(43)(44)(45)(46). Psoralen monoadducts are somewhat helix-stabilizing, whereas crosslinks are helix-destabilizing (47,48).…”

Section: Discussionmentioning

confidence: 99%

Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions

Vásquez

Christensen

et al. 2002

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

111

128

View full text Add to dashboard Cite

Nucleotide excision repair (NER) plays a central role in maintaining genomic integrity by detecting and repairing a wide variety of DNA lesions. Xeroderma pigmentosum complementation group A protein (XPA) is an essential component of the repair machinery, and it is thought to be involved in the initial step as a DNA damage recognition and͞or confirmation factor. Human replication protein A (RPA) and XPA have been reported to interact to form a DNA damage recognition complex with greater specificity for damaged DNA than XPA alone. The mechanism by which these two proteins recognize such a wide array of structures resulting from different types of DNA damage is not known. One possibility is that they recognize a common feature of the lesions, such as distortions of the helical backbone. We have tested this idea by determining whether human XPA and RPA proteins can recognize the helical distortions induced by a DNA triple helix, a noncanonical DNA structure that has been shown to induce DNA repair, mutagenesis, and recombination. We measured binding of XPA and RPA, together or separately, to substrates containing triplexes with three, two, or no strands covalently linked by psoralen conjugation and photoaddition. We found that RPA alone recognizes all covalent triplex structures, but also forms multivalent nonspecific DNA aggregates at higher concentrations. XPA by itself does not recognize the substrates, but it binds them in the presence of RPA. Addition of XPA decreases the nonspecific DNA aggregate formation. These results support the hypothesis that the NER machinery is targeted to helical distortions and demonstrate that RPA can recognize damaged DNA even without XPA.triple helix ͉ nucleotide excision repair B ecause genome stability is critical for cell survival, extremely sensitive DNA repair mechanisms have evolved to protect the genome from both internal and external assaults. Defects in these repair mechanisms can lead to severe disorders such as xeroderma pigmentosum, ataxia telangiectasia, Fanconi anemia, and Bloom syndrome (reviewed in refs. 1-5). One of the major consequences of these defects is an enhanced predisposition to cancer. It has been reported that 80-90% of human cancers are the result of DNA damage (6).Many types of DNA damage, spontaneous and induced, develop from both endogenous and exogenous sources. Exogenous damages, both chemical and physical, arise from exposure to UV and ionizing radiation and from natural and synthetic chemicals. Cellular metabolic processes lead to internal sources of DNA damage; for example, it is estimated that loss of a purine base (depurination) occurs at a rate of nearly 20,000 events per cell per day (7).In humans, different types of DNA damage are thought to be repaired by one of several mechanisms, including nucleotide excision repair (NER; reviewed in refs. 8 and 9). NER removes covalent DNA lesions and is the only known mechanism for removing bulky DNA adducts in humans. The damaged base is removed by endonucleolytic cleavage of the phosphodiester bond...

show abstract

Section: Discussionmentioning

confidence: 99%

Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions

Vásquez

Christensen

et al. 2002

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

111

128

View full text Add to dashboard Cite

show abstract

“…): CRL1589 (AA8 wild type), CRL1867 (UV135, XPG mutant), and irs1SF (XRCC3 mutant). Cell extracts were prepared as described previously (30). Recombinant XPF-ERCC1 was purified as described previously (2).…”

Section: Methodsmentioning

confidence: 99%

DNA Interstrand Cross-Links Induce Futile Repair Synthesis in Mammalian Cell Extracts

Bessho

Nechev³

et al. 2000

Molecular and Cellular Biology

Self Cite

124

121

View full text Add to dashboard Cite

DNA interstrand cross-links are induced by many carcinogens and anticancer drugs. It was previously shown that mammalian DNA excision repair nuclease makes dual incisions 5 to the cross-linked base of a psoralen cross-link, generating a gap of 22 to 28 nucleotides adjacent to the cross-link. We wished to find the fates of the gap and the cross-link in this complex structure under conditions conducive to repair synthesis, using cell extracts from wild-type and cross-linker-sensitive mutant cell lines. We found that the extracts from both types of strains filled in the gap but were severely defective in ligating the resulting nick and incapable of removing the cross-link. The net result was a futile damage-induced DNA synthesis which converted a gap into a nick without removing the damage. In addition, in this study, we showed that the structure-specific endonuclease, the XPF-ERCC1 heterodimer, acted as a 3-to-5 exonuclease on cross-linked DNA in the presence of RPA. Collectively, these observations shed some light on the cellular processing of DNA cross-links and reveal that cross-links induce a futile DNA synthesis cycle that may constitute a signal for specific cellular responses to cross-linked DNA.Interstrand cross-links are common lesions introduced into DNA by drugs such as psoralen, cisplatin, mitomycin C, and melphalan (17). These lesions are eliminated from DNA by a mechanism involving excision repair and recombination in Escherichia coli (3,5,32) and in yeast (15,20). In mammalian cells, the precise role of excision repair in eliminating crosslinks is not known (35,36). Although mutations in any of the genes required for the dual-incision step of excision repair cause sensitivity to cross-linking chemicals, the XPF and ERCC1 mutant cell lines, in addition to being defective in excision repair, are particularly sensitive to cross-linking agents and hence have been presumed to play a special role in crosslink repair (13). Similarly, mutations in the XRCC2 and XRCC3 genes, encoding proteins with sequence homology to the human RAD51 protein (19), confer sensitivity to crosslinking agents without affecting the excision repair system and hence are thought to play a unique role in processing of crosslinks (35). To understand the mechanism of cross-link repair, it appears that the actions of the excision repair system, the XPF-ERCC1 complex, and XRCC2 and XRCC3 on crosslinks must be investigated.The human nucleotide excision repair system removes base monoadducts and intrastrand diadducts by making a dual incision bracketing the lesion (14). Recently, we reported the surprising finding that with a cross-linked substrate, the human excision nuclease makes both incisions 5Ј to the cross-linked base, excising a damage-free oligomer and generating a gap of 22 to 28 nucleotides (nt) 5Ј to either the furan-side or the pyrone-side adducted thymine of a psoralen cross-link (1). We proposed that the gap generated by this unusual type of dual incision may initiate at least one pathway of cross-link repair. In the present...

show abstract

“…UVA photoactivation was used to generate a mixture of targeted monoadducts and cross-links (12), consisting of the psoralen-oligonucleotides covalently linked to either one strand of the target duplex or to both strands of the duplex, respectively. The damaged plasmids were incubated in HeLa whole cell extracts (24,34,35). The products were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography.…”

Section: Figmentioning

confidence: 99%

Altered Repair of Targeted Psoralen Photoadducts in the Context of an Oligonucleotide-mediated Triple Helix

Wang

Glazer

1995

Journal of Biological Chemistry

View full text Add to dashboard Cite

Oligonucleotides can bind as third strands of DNA in a sequence-specific manner to form triple helices. Psoralen-conjugated, triplex-forming oligonucleotides (TFOs) have been used for the site-specific modification of DNA to inhibit transcription and to target mutations to selected genes. Such strategies, however, must take into account the ability of the cell to repair the triplexdirected lesion. We report experiments showing that the pattern of mutations produced by triplex-targeted psoralen adducts in an SV40 shuttle vector in monkey COS cells can be influenced by the associated third strand. Mutations induced by psoralen adducts in the context of a TFO of length 10 were the same as those generated by isolated adducts but were found to be different from those generated in the presence of a TFO of length 30 at the same target site. In complementary experiments, HeLa whole cell extracts were used to directly assess repair of the TFO-directed psoralen adducts in vitro. Excision of the damaged DNA was inhibited in the context of the 30-mer TFO, but not the 10-mer. These results suggest that an extended triple helix of length 30, which exceeds the typical size of the nucleotide excision repair patch in mammalian cells, can alter repair of an associated psoralen adduct. We present a model correlating these results and proposing that the incision steps in nucleotide excision repair in mammalian cells can be blocked by the presence of a third strand of sufficient length and binding affinity, thereby changing the pattern of mutations. These results may have implications for the use of triplex-forming oligonucleotides for genetic manipulation, and they may lead to the use of such oligonucleotides as tools to probe DNA repair pathways.Oligonucleotides can bind in the major groove of duplex DNA to form triple helices in a sequence-specific manner (1-4). Progress in elucidating the third strand binding code has raised the possibility of developing nucleic acids as sequencespecific reagents for research and possibly therapeutic applications. Oligonucleotide-mediated triplex formation has been shown to inhibit transcription factor binding to promoter sites and to block transcription in vitro and in vivo (5). Strategies to enhance transcription inhibition using oligonucleotide-intercalator conjugates have also been employed (6, 7). Triplex formation has further been used as a tool to generate unique cleavage sites in DNA in vitro (8). We and others have explored the use of triplex-forming oligonucleotides (TFOs) 1 as a method to deliver a tethered mutagen to a selected gene for the site-specific introduction of DNA damage (9 -12). Psoralen-conjugated oligonucleotides directed to a site in a mutation reporter gene were found to induce targeted mutations in (10) and SV40 (9) DNA in experiments in which the triplex formation was carried out in vitro. In recent work, conditions were determined under which targeted mutagenesis of an SV40 vector could be achieved in vivo via intracellular triplex formation using psoralen-linked TF...

show abstract

Removal of psoralen monoadducts and crosslinks by human cell free extracts

Cited by 43 publications

References 22 publications

Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions

Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions

DNA Interstrand Cross-Links Induce Futile Repair Synthesis in Mammalian Cell Extracts

Altered Repair of Targeted Psoralen Photoadducts in the Context of an Oligonucleotide-mediated Triple Helix

Contact Info

Product

Resources

About