The fate of 8-methoxypsoralen photoinduced crosslinks in nuclear and mitochondrial yeast DNA: comparison of wild-type and repair-deficient strains.

Abstract

In, Saccharomyce8 cerevisiae, after 8-methoxypsoralen 18-(OMe)Ps] photoaddition, more crosslinks are induced per unit dose in mitochondrial DNA than in nuclear DNA. In wildtype cells treated.in the exponential phase of growth, single-and double-strand breaks are produced during crosslink removal and then are rejoined upon postexposure incubation. The incision step is almost blocked in the rad3-2 mutant, which is also defective in excision-repair of UV-induced (254 nm) pyrimidine dimers. The cutting of crosslinks from nuclear DNA is depressed in wild-type stationary-phase cells. This is correlated with a higher sensitivity of such cells to 8-(OMe)Ps photoinduced, cell killing. The incision of crosslinks is dramatically reduced in mitochondrial DNA. The rejoining ofsingle-and double-strand breaks is not only dependent on the product of the RAD51 gene (as shown by others) but also of the PS02 gene. A correlation was found.between the ability to recombine and strand rejoining. Therefore, as in bacteria, both the excision and the recombinational repair systems are. involved in crosslink repair in yeast. However, double-strand breaks in yeast constitute repair intermediates which are not detected in Eacherichia coli. The LD37 (dose necessary to induce a mean of one lethal hit per cell) corresponds to about 120 crosslinks per genome in.exponential-phase cells of the wild type and to 1-2 crosslinks in the p8o2-1 mutant.The psoralen derivative, 8-methoxypsoralen [8-(OMe)Ps] intercalates in DNA; upon exposure to near UV radiation, it reacts with pyrimidine bases to form monoadducts and interstrand crosslinks (1, 2). These photomodifications of DNA lead to lethality and induction of mutation and recombination (1-3). In addition, the production in yeast of the mitochondrial "petite" mutation (rho-) has been reported (4-6). Removal ofcrosslinks has been described in Escherichia coli (7-10) and in mammalian cells in culture (11,12). In E. coli, the excision and the recombinational repair pathways are involved in the restitution of an intact genome from DNA containing psoralen photoproducts (7,9,10). In eukaryotes, limited information is available on the molecular steps in relation to the genetic control of repair of crosslinks in DNA. On the basis of sensitivity to cell killing in rad mutants compared to the wild type, it was demonstrated that in the yeast Saccharomyces cerevisiae, in addition to the excision (rad3 type), the error-prone (rad6 type), and the DNA double-strand-break (rad52) repair pathways (5, 13), some repair steps controlled by a class of genes designated pso (14) also are implicated in the repair of this last type of lesion.In this report, we determined the proportion of 8-(OMe)Ps photoinduced crosslinks as a function ofdose and followed their fate in nuclear and mtDNA in a normal strain. The influence of the growth phases, known to modify the lethal response (15), on crosslink removal was studied. An excision-deficient mutant (rad3) and one of the pso mutants (pso2) defective in the G2-phase repai...

Human SNM1B is required for normal cellular response to both DNA interstrand crosslink-inducing agents and ionizing radiation

“…The appearance of DSB as an intermediate in the ICL repair has been documented for S. cerevisiae (Jachymczyk et al, 1981;Magana-Schwencke et al, 1982;McHugh et al, 2000), and there is clear evidence that DSBs occur during ICL repair in mammalian cells as well (De Silva et al, 2000;Rothfuss and Grompe, 2004). hSNM1C/ ARTEMIS functions in the NHEJ pathway of DSB repair, where it complexes with DNA-PK CS and processes 5 0 and 3 0 overhangs (Ma et al, 2002).…”

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

“…In yeast cells the formation of psoralen-induced DSBs depends upon NER function (2,5,19,20). However, NER is dispensable for the generation of DSBs by nitrogen mustard cross-links in both yeast and mammalian cells (6,23).…”

“…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). Rejoining of the cross-link-induced DSBs depends on the HR genes RAD51 and RAD52 (2,5,(21)(22)(23). These observations suggest the following two-step model for psoralen cross-link repair; 1) NER acts on cross-links to produce DSBs, and 2) the DSBs are repaired by HR.…”

The nucleotide sequence of theRAD3gene ofSaccharomyces cerevisiae: a potential adenine nucleotide binding amino acid sequence and a nonessential acidic carboxyl terminal region

“…A mutation in any of the first six of the ten genes listed results in highly defective incision of DNA containing pyrimidine dimers (13,14) or interstrand DNA crosslinks (8,11,12), while a mutation in RAD14, produces reduced incision proficiency compared with the Rad+ strain (9,13). The RAD3 gene has been cloned and partially characterized (15,16).…”

“…In the yeast Saccharomyces cerevisiae, excision of pyrimidine dimers or interstrand DNA crosslinks requires a large number of genes -RADI, RAD2, RAD3, RAD4, RAD1O, MMS19, RAD7, RAD14, RAD16, and RAD23 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). A mutation in any of the first six of the ten genes listed results in highly defective incision of DNA containing pyrimidine dimers (13,14) or interstrand DNA crosslinks (8,11,12), while a mutation in RAD14, produces reduced incision proficiency compared with the Rad+ strain (9,13).…”