Structure of the DNA interstrand crosslink of 4,5',8-trimethylpsoralen

Previously, we have demonstrated that human oxidative DNA glycosylase NEIL1 excises photoactivated psoralen-induced monoadducts but not genuine interstrand cross-links (ICLs) in duplex DNA. It has been postulated that the repair of ICLs in mammalian cells is mainly linked to DNA replication and proceeds via dual incisions in one DNA strand that bracket the cross-linked site. This process, known as "unhooking," enables strand separation and translesion DNA synthesis through the gap, yielding a threestranded DNA repair intermediate composed of a short unhooked oligomer covalently bound to the duplex. At present, the detailed molecular mechanism of ICL repair in mammalian cells remains unclear. Here, we constructed and characterized three-stranded DNA structures containing a single ICL as substrates for the base excision repair proteins. We show that NEIL1 excises with high efficiency the unhooked ICL fragment within a three-stranded DNA structure. Complete reconstitution of the repair of unhooked ICL shows that it can be processed in a short patch base excision repair pathway. The new substrate specificity of NEIL1 points to a preferential involvement in the replication-associated repair of ICLs. Based on these data, we propose a model for the mechanism of ICL repair in mammalian cells that implicates the DNA glycosylase activity of NEIL1 downstream of Xeroderma Pigmentosum group F/Excision Repair Cross-Complementing 1 endonuclease complex (XPF/ERCC1) and translesion DNA synthesis repair steps. Finally, our data demonstrate that Nei-like proteins from Escherichia coli to human cells can excise bulky unhooked psoralen-induced ICLs via hydrolysis of glycosidic bond between crosslinked base and deoxyribose sugar, thus providing an alternative heuristic solution for the removal of complex DNA lesions.Interstrand cross-links (ICLs) 4 are highly lethal DNA lesions that block DNA transcription, replication, and recombination by preventing strand separation. Due to their high cytotoxicity, DNA cross-linking agents such as mitomycin C, cisplatin, and psoralens are widely used against hyperplasic diseases such as cancer and psoriasis (1, 2). Furanocoumarins (psoralens), naturally occurring secondary metabolites in plants, are tricyclic compounds formed by the fusion of a furan ring with a coumarin (Fig. 1). Among other ICL-inducing agents, psoralens require UVA photoactivation following DNA intercalation to chemically react with both cellular DNA in vivo and naked DNA in vitro (3). 8-Methoxypsoralen (8-MOP) is an asymmetric, planar compound that intercalates into DNA duplex near pyrimidines, preferentially at 5Ј-TpA sites. Upon photoactivation, 8-MOP primarily photoalkylates DNA by cycloaddition to the 5,6-double bond of a thymidine generating monoadducts (MA) with either the 4Ј,5Ј-double bond of the furan (MAf) or the 3,4-double bond of the pyrone (MAp) side of the psoralen (4) (supplemental Fig. S1). A unique property of psoralen photochemistry is that the absorption of a second photon by the MAf leads to formation of a p...

Section: Construction and Characterization Of The Three-stranded Dna supporting

confidence: 62%

“…The resulting oligonucleotide duplexes were used to generate MAs and ICLs as described (23). It should be emphasized that all psoralen-induced MAs used in this work were pyrone-sided (MAp) and obtained by incubation of purified cross-linked duplex in alkali as described (25).…”

Section: Methodsmentioning

confidence: 99%

The Human Oxidative DNA Glycosylase NEIL1 Excises Psoralen-induced Interstrand DNA Cross-links in a Three-stranded DNA Structure

Couvé

Macé-Aimé

Rosselli

et al. 2009

“…The transplatin ICL structure results in no extrahelical cytosines, whereas in the oxaliplatin ICL structure, the flanking cytosines are not as exposed as in cisplatin ICLs and protein recognition is altered (45)(46)(47). The ICL structure is also distinct from those formed by mitomycin C and psoralen, and down-regulation of BER proteins resulted in hypersensitivity to these cross-linking agents (48,49). These structural differences suggest that cisplatin could elicit different responses than other cross-linking agents.…”

Section: Discussionmentioning

confidence: 99%

Novel Role of Base Excision Repair in Mediating Cisplatin Cytotoxicity

Kothandapani

Dangeti

Brown

et al. 2011

121

Using isogenic mouse embryonic fibroblasts and human cancer cell lines, we show that cells defective in base excision repair (BER) display a cisplatin-specific resistant phenotype. This was accompanied by enhanced repair of cisplatin interstrand crosslinks (ICLs) and ICL-induced DNA double strand breaks, but not intrastrand adducts. Cisplatin induces abasic sites with a reduced accumulation in uracil DNA glycosylase (UNG) null cells. We show that cytosines that flank the cisplatin ICLs undergo preferential oxidative deamination in vitro, and AP endonuclease 1 (APE1) can cleave the resulting ICL DNA substrate following removal of the flanking uracil. We also show that DNA polymerase ␤ has low fidelity at the cisplatin ICL site after APE1 incision. Down-regulating ERCC1-XPF in BER-deficient cells restored cisplatin sensitivity. Based on our results, we propose a novel model in which BER plays a positive role in maintaining cisplatin cytotoxicity by competing with the productive cisplatin ICL DNA repair pathways.

“…The distance between the 3Ј and 5Ј incisions for furan side monoadducts and cross-links was always 9 nucleotides, whereas the distance between these incisions for the pyrone side monoadducts and cross-links was always 17 nucleotides. This increased distance between sites of incision for the pyrone versus the furan side adducts could be due to the fact that, since TMP is an asymmetric molecule, more distortion may occur in the DNA in the vicinity of the thymine adducted to the pyrone side of TMP as compared with that of the thymine bound to the furan side (7,9,10,37). The sites of incision on the 3Ј side of both types of furan side and pyrone side adducts were similar; they were either at the fourth or fifth phosphodiester bond from the adducted thymine, depending upon the adduct (Fig.…”

Section: Construction Of a 132-bp Dna Substrate Containing Sitespecifmentioning

confidence: 99%

“…Psoralen-DNA adducts are formed in three stages; psoralen first intercalates into the DNA duplex in a noncovalent manner and then, upon photoreaction with UVA light, forms either a furan side or a pyrone side monoadduct with the 5,6-double bond of a pyrimidine, which the majority of the time is a thymine. If the furan side of the molecule is linked to DNA, then further exposure to UVA light leads to production of an interstrand cross-link (5)(6)(7)(8)(9)(10).…”

mentioning

confidence: 99%

Human Endonucleolytic Incision of DNA 3′ and 5′ to a Site-directed Psoralen Monoadduct and Interstrand Cross-link

Kumaresan¹,

Hang

Lambert³

1995

Self Cite

Human chromatin-associated protein extracts were examined for endonucleolytic activity on a defined 132-base pair DNA substrate containing a single, site-specific 4,5,8-trimethylpsoralen plus long wavelength ultraviolet light-induced furan side or pyrone side monoadduct or interstrand cross-link. These extracts produced incisions on both the 3 and 5 sides of each of these lesions. The distance between the 3 and 5 incisions at sites of a furan side monoadduct or cross-link was 9 nucleotides, and at sites of a pyrone side monoadduct or cross-link it was 17 nucleotides. Incisions on the 3 side of both types of furan side and pyrone side adducts were similar and were either at the fourth or fifth phosphodiester bond from the adducted thymine, depending upon the adduct. However, greater differences were observed between sites of 5 incision. This incision occurred at the fifth and sixth phosphodiester bonds from the adducted thymine at sites of furan side monoadducts and cross-links, respectively, and at the 13th and 14th phosphodiester bonds at sites of pyrone side monoadducts and cross-links, respectively. Thus, direct analysis of sites of endonucleolytic incision reveals that the location of sites of incision on TMP-adducted substrates depends upon the type of adduct present.Repair of DNA interstrand cross-links is critical for a number of cellular processes such as transcription and DNA replication and therefore is particularly important for the maintenance of genetic integrity and cellular survival. A nucleotide excision repair mechanism is responsible in both prokaryotes and eukaryotes for the removal of this type of lesion (1-3). Although the molecular basis of repair of DNA interstrand cross-links has been extensively studied in bacteria, the proteins involved in the removal of these lesions in mammalian cells and their mechanism of action is largely unknown. A critical, rate-limiting step in this repair process is the initial damage recognition and incision step in which a protein specifically locates or recognizes a site of damage and an endonuclease makes an incision on the DNA strand at or near this site. We have recently identified a damage recognition protein in normal human chromatin, which binds to DNA containing interstrand cross-links produced by 4,5Ј,8-trimethylpsoralen (TMP) 1 plus long wavelength ultraviolet (UVA) light, and our studies suggest that it plays a role in the repair of interstrand cross-links (4). However, an endonuclease that specifically incises DNA at sites of these interstrand cross-links has heretofore not been isolated from mammalian cells.A number of different agents have been shown to produce interstrand cross-links in DNA. One of the most definitive of these, whose reaction with DNA has been well characterized, is psoralen plus UVA light. Psoralens are a group of three-ring heterocyclic furocoumarins that contain two reactive double bonds, a 4Ј,5Ј-double bond in the furan ring and a 3,4-double bond in the pyrone ring (5-8). Psoralen-DNA adducts are formed in three stages; pso...