Psoralens as Photoactive Probes of Nucleic Acid Structure and Function: Organic Chemistry, Photochemistry, and Biochemistry

Cimino, George D.; Gamper, Howard; Isaacs, Stephen T.; Hearst, John E.

doi:10.1146/annurev.bi.54.070185.005443

Cited by 750 publications

(376 citation statements)

References 124 publications

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“…Psoralens are tricyclic furocumarins that intercalate between the two strands of the DNA double helix and photoreact to form cyclobutane adducts with pyrimidine bases. Irradiation of DNA containing intercalated psoralen with longwave UV light (365 nm) results in the formation of monoadducts and diadducts by which the two strands of the DNA double helix become covalently joined through a psoralen molecule (20). For RIs, we anticipated that the long-wave UV-light-induced crosslinks would prevent separation of nascent strands from their corresponding parentals, avoiding the formation of HLJs and FR.…”

Section: Resultsmentioning

confidence: 99%

“…At elevated concentrations of EthBr, however, these RIs regain electrophoretic mobility, suggesting they have acquired (ϩ) ⌬Wr. In an attempt to solve this apparent controversy, here we used 2D agarose gel electrophoresis (18,19), electron microscopy, and psoralen cross-linking (20) to reinvestigate the topological behavior of the RIs of pBR18-TerE@StyI exposed to high concentrations of an intercalanting agent. pBR18-TerE@StyI is a ColE1 derivative of pBR18 (21) containing the E. coli replication terminator TerE (22,23) cloned at the unique StyI site of the plasmid (11).…”

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Topological locking restrains replication fork reversal

Fierro-Fernández

Hernández

Krimer

et al. 2007

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

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Two-dimensional agarose gel electrophoresis, psoralen crosslinking, and electron microscopy were used to study the effects of positive supercoiling on fork reversal in isolated replication intermediates of bacterial DNA plasmids. The results obtained demonstrate that the formation of Holliday-like junctions at both forks of a replication bubble creates a topological constraint that prevents further regression of the forks. We propose that this topological locking of replication intermediates provides a biological safety mechanism that protects DNA molecules against extensive fork reversals.DNA repair ͉ DNA replication ͉ DNA structure ͉ DNA topology ͉ DNA supercoiling G enomic instability is a hallmark of several human diseases (1, 2). During the last decade it was clearly established that a significant portion of this instability arises during the replication of damaged templates. Progression of the replication machinery halts or pauses when the replisome encounters DNA lesions, and this blockage may lead to their collapse (3). All living cells are constantly exposed to a great variety of genotoxic agents. Among these agents, UV light and tobacco smoke, because of their well known carcinogenic potential, are particularly important for humans (4). This correlation explains the increasing interest of the scientific community to understand the mechanisms that cells have developed to repair DNA lesions and reactivate stalled replication forks to ensure that when a cell divides both daughter cells receive a complete and exact copy of the genome of their mother cell (5).In Escherichia coli, current models suggest that once a replication fork meets a DNA lesion RecA (6) and/or RecG (5, 7) promotes regression of the stalled fork, generating a Hollidaylike junction (HLJ) that is subsequently processed by the Ruv-ABC complex to allow replication restart by PriA (8-10). Despite the solid genetic evidence supporting this model, the molecular characterization of HLJs formed in vivo and an estimation of the extent of fork regression are still scarce (11-13). Moreover, replication restart after fork stalling was also suggested to occur by means of mechanisms that do not necessarily need fork reversal (FR) (8,14).One of the possible driving forces for FR at stalled forks in vivo is the positive (ϩ) writhe increase (⌬Wr) generated by other replication or transcription forks progressing in the opposite direction (15-17). FR relaxes this (ϩ) ⌬Wr and lowers the free energy of the constrained DNA. Indeed, in vitro experiments demonstrated that FR compensates the (ϩ) ⌬Wr generated by intercalation of moderate quantities of chloroquine in the unreplicated region of replication intermediates (RIs) (16). In the same study, however, direct scanning force microscopy of RIs exposed to rather high concentrations of ethidium bromide (EthBr) revealed their supercoiled character, suggesting that FR was apparently unable to adsorb all of the (ϩ) ⌬Wr generated by the intercalated drug. In a different study, Olavarrieta et al. (11) also noticed t...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Topological locking restrains replication fork reversal

Fierro-Fernández

Hernández

Krimer

et al. 2007

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

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“…Psoralen undergoes photoreaction with DNA to form monoadducts and interstrand cross-links (15,16). Psoralen photoreaction of replicating yeast cells induces NER-dependent DNA breaks; monoadducts induce single-strand nicks, whereas interstrand cross-links induce DSBs (2,5,(17)(18)(19)(20).…”

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DNA Repair Defects Channel Interstrand DNA Cross-links into Alternate Recombinational and Error-prone Repair Pathways

Saffran

Ahmed

Bellevue

et al. 2004

Journal of Biological Chemistry

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The repair of psoralen interstrand cross-links in the yeast Saccharomyces cerevisiae involves the DNA repair groups nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (PRR). In repair-proficient yeast cells cross-links induce double-strand breaks, in an NER-dependent process; the double-strand breaks are then repaired by HR. An alternate error-prone repair pathway generates mutations at cross-link sites. We have characterized the repair of plasmid molecules carrying a single psoralen cross-link, psoralen monoadduct, or double-strand break in yeast cells with deficiencies in NER, HR, or PRR genes, measuring the repair efficiencies and the levels of gene conversions, crossing over, and mutations. Strains with deficiencies in the NER genes RAD1, RAD3, RAD4, and RAD10 had low levels of cross-link-induced recombination but higher mutation frequencies than repair-proficient cells. Deletion of the HR genes RAD51, RAD52, RAD54, RAD55, and RAD57 also decreased induced recombination and increased mutation frequencies above those of NER-deficient yeast. Strains lacking the PRR genes RAD5, RAD6, and RAD18 did not have any crosslink-induced mutations but showed increased levels of recombination; rad5 and rad6 cells also had altered patterns of cross-link-induced gene conversion in comparison with repair-proficient yeast. Our observations suggest that psoralen cross-links can be repaired by three pathways: an error-free recombinational pathway requiring NER and HR and two PRR-dependent errorprone pathways, one NER-dependent and one NERindependent.DNA interstrand cross-links are complex lesions, highly toxic to cells, and difficult to repair because of the involvement of both strands of the DNA duplex. A single unrepaired interstrand cross-link is sufficient to kill a cell, and multiple DNA repair pathways may be required to complete cross-link removal (1-7).The yeast Saccharomyces cerevisiae has three major DNA repair epistasis groups, all of which are involved in cross-link repair; they are nucleotide excision repair, recombinational repair, and post-replication repair (8). Nucleotide excision repair (NER) 1 is a general system that recognizes bulky and helix-distorting lesions (9, 10). Endonucleases nick the affected DNA strand on both the 5Ј and 3Ј sides of the lesion; after removal of the damaged oligonucleotide, the resulting singlestrand gap is filled in by polymerase activity using the undamaged strand as a template. This mechanism produces error-free repair of single-strand damage. Recombinational repair is the major pathway for repair of double-strand damage, such as double-strand breaks (DSBs) or gaps in yeast (11,12). In homologous recombination (HR) the broken DNA molecule is repaired, and missing genetic information is restored through interactions with intact homologous sequences. This pathway is also non-mutagenic but can result in DNA rearrangements. Post-replication repair (PRR) acts on damage in the context of DNA replication, allowing for bypass of replication fork-...

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“…15-17) for reviews). Upon exposure to long wavelength UV light (UVA; 310 -400 nm), 1 psoralens react with DNA or RNA to form covalent adducts (12,17,18). In DNA, psoralens react primarily with thymidine and to a lesser extent with cytosine.…”

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Cross-linking of DNA-binding Proteins to DNA with Psoralen and Psoralen Furan-side Monoadducts

Sastry¹,

Ross²,

Parraga³

1997

Journal of Biological Chemistry

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We have developed a novel photocross-linking technique using free 8-methoxypsoralen and DNA furan-side monoadducts plus long wave ultraviolet light (UVA). Both sequence-specific (Max) and nonspecific (RecA and T7 RNA polymerase) DNA-binding proteins were crosslinked. The macroscopic equilibrium binding constant (ϳ10 9 M ؊1 ) and DNase I footprinting indicated that binding of Max to its cognate sequence (E-box) was unimpaired by 8-methoxypsoralen and that cross-linking occurred in normal complexes. RecA protein and T7 RNA polymerase were cross-linked to a 12-mer DNA furanside monoadduct with UVA. Cross-link yields were directly proportional to the UVA dose. Cross-links were stable to 8 M urea, 1-10% SDS, commonly used alcohols, and mild acids (5% trichloroacetic acid). The DNA in cross-links was reversed with 254 nm UV (photoreversal) or with hot base (base-catalyzed reversal), consistent with (2 ؉ 2) cycloaddition via the 4 ,5 -furan of the psoralen. Comparative action spectra for DNA-DNA cross-linking and DNA-protein cross-linking revealed that the latter occurred maximally at 300 nm, while the former occurred maximally at 320 nm. This 20-nm blue shift suggested a higher potential energy surface for an excited psoralen participating in protein-DNA crosslinking as compared with DNA-DNA cross-linking. As with DNA-DNA cross-linking, DNA-protein cross-linking is a two-photon process. Absorption of the first photon formed a 4 ,5 -adduct with DNA, which then absorbed a second photon, leading to cross-linking to protein. Based on the action spectra and the known excited states of psoralen, it is suggested that the triplet n, * transition localized in the C-2‫؍‬O of psoralen may be involved in protein-psoralen photoreactions.

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Psoralens as Photoactive Probes of Nucleic Acid Structure and Function: Organic Chemistry, Photochemistry, and Biochemistry

Cited by 750 publications

References 124 publications

Topological locking restrains replication fork reversal

Topological locking restrains replication fork reversal

DNA Repair Defects Channel Interstrand DNA Cross-links into Alternate Recombinational and Error-prone Repair Pathways

Cross-linking of DNA-binding Proteins to DNA with Psoralen and Psoralen Furan-side Monoadducts

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