Dietrich Averbeck

Reassembly of shattered chromosomes in Deinococcus radiodurans.

Dehydration or desiccation is one of the most frequent and severe challenges to living cells. The bacterium Deinococcus radiodurans is the best known extremophile among the few organisms that can survive extremely high exposures to desiccation and ionizing radiation, which shatter its genome into hundreds of short DNA fragments. Remarkably, these fragments are readily reassembled into a functional 3.28-megabase genome. Here we describe the relevant two-stage DNA repair process, which involves a previously unknown molecular mechanism for fragment reassembly called ‘extended synthesis-dependent strand annealing’ (ESDSA), followed and completed by crossovers. At least two genome copies and random DNA breakage are requirements for effective ESDSA. In ESDSA, chromosomal fragments with overlapping homologies are used both as primers and as templates for massive synthesis of complementary single strands, as occurs in a single-round multiplex polymerase chain reaction. This synthesis depends on DNA polymerase I and incorporates more nucleotides than does normal replication in intact cells. Newly synthesized complementary single-stranded extensions become ‘sticky ends’ that anneal with high precision, joining together contiguous DNA fragments into long, linear, double-stranded intermediates. These intermediates require RecA-dependent crossovers to mature into circular chromosomes that comprise double-stranded patchworks of numerous DNA blocks synthesized before radiation, connected by DNA blocks synthesized after radiation.

Characterization of homologous recombination induced by replication inhibition in mammalian cells.

To analyze relationships between replication and homologous recombination in mammalian cells, we used replication inhibitors to treat mouse and hamster cell lines containing tandem repeat recombination substrates. In the first step, few double‐strand breaks (DSBs) are produced, recombination is slightly increased, but cell lines defective in non‐homologous end‐joining (NHEJ) affected in ku86 (xrs6) or xrcc4 (XR‐1) genes show enhanced sensitivity to replication inhibitors. In the second step, replication inhibition leads to coordinated kinetics of DSB accumulation, Rad51 foci formation and RAD51‐dependent gene conversion stimulation. In xrs6 as well as XR‐1 cell lines, Rad51 foci accumulate more rapidly compared with their respective controls. We propose that replication inhibition produces DSBs, which are first processed by the NHEJ; then, following DSB accumulation, RAD51 recombination can act.

Recent advances in psoralen phototoxicity mechanism

The present overview outlines some of the recent research developments leading to a better understanding of the mechanisms which underlie psoralen induced phototoxicity

Photochemotherapy (PUVA) of psoriasis using 3-carbethoxypsoralen, a non-carcinogenic compound in mice.

The carcinogenic risk of photochemotherapy (PUVA) with bi‐functional furocoumarins such as 8‐methoxypsoralen (8‐MOP) which form cross‐links in cellular DNA has initiated a search for active but less hazardous psoralens. A new compound, 3‐carbethoxypsoralen (3‐CPs), studied in the yeast Saccharomyces cerevisiae (eukaryote), has been shown to be very photoactive on DNA and to form only mono‐additions to DNA. These lesions appear to be more easily repaired than the cross‐links induced by 8‐MOP. 3‐CPs produces less nuclear genetic events such as nuclear mutations and mitotic crossovers, but more cytoplasmic ‘petite’ mutations (damage to mitochondrial DNA) than 8‐MOP. In mice it was demonstrated that after local or intra‐peritoneal administration, in contrast to 8‐MOP. 3‐CPs is non‐toxic, non‐erythematogenic, and non‐carcinogenic. A study of ten psoriatic patients has shown that local applications of 3‐CPs plus UV‐A exhibit about the same therapeutic activity for the clearing of psoriatic lesions as local treatment with 8‐MOP plus UV‐A, but without any localized hyperpigmentation.

The debate on the use of linear no threshold for assessing the effects of low doses.

From December 2004 to July 2005, three reports on the effects of low doses of ionising radiation were released: ICRP (2004), the joint report of the French Academies of Science and Medicine (Tubiana et al 2005), and a report from the American Academy of Sciences (BEIR VII 2005). These reports quote the same recent articles on the biological effects of low doses, yet their conclusions diverge. The French report concludes that recent biological data show that the efficacy of defense mechanisms is modulated by dose and dose rate and that linear no threshold (LNT) is no longer plausible. The ICRP and the BEIR VII reports recognise that there are biologic arguments against LNT but feel that there are not sufficient biological proofs against it to change risk assessment methodology and subsequent regulatory policy based on LNT. They point out the remaining uncertainties and the lack of mechanistic explanations of phenomena such as low dose hyperlethality or the adaptive response. In this context, a critical analysis of the available data is necessary. The epidemiological data and the experimental data challenge the validity of the LNT hypothesis for assessing the carcinogenic effect of low doses, but do not allow its exclusion. Therefore, the main criteria for selecting the most reliable dose-effect relationship from a scientific point of view should be based on biological data. Their analysis should help one to understand the current controversy.

Relationship between lesions photoinduced by mono- and bi-functional furocoumarins in DNA and genotoxic effects in diploid yeast

The induction of genetic effects was studied in a diploid strain of Saccharomyces cerevisiae (D7) after treatments with the monofunctional furocoumarins 7-methylpyrido[3,4-c]psoralen (MePyPs), pyrido[3,4-c]psoralen (PyPs) and 3-carbethoxypsoralen (3-CPs) and the bifunctional furocoumarins 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) in the presence of 365-nm radiation. The DNA photobinding of radioactively labelled MePyPs, 3-CPs, 5-MOP and 8-MOP was determined in parallel. The DNA-photobinding capacity was highest for MePyPs followed in decreasing order by 5-MOP, 3-CPs and 8-MOP. At a concentration of 5 microM and 4.2 kJ/m2 of 365-nm radiation approximately 160, 66, 60 and 16 adducts per 10(6) base pairs were formed by MePyPs, 5-MOP, 3-CPs and 8-MOP, respectively. The activity of MePyPs and PyPs for the induction of lethal effects lay in the same range as that of 5-MOP whereas 8-MOP was 3 times less active and 3-CPs showed very little activity. For the induction of mitotic gene conversion and genetically altered colonies including mitotic crossing-over the order of activity was about the same as that observed for the induction of lethal effects: MePyPs greater than 5-MOP greater than PyPs greater than 8-MOP much greater than 3-CPs. Nuclear reversions were induced most effectively by 5-MOP, 8-MOP being about 3 times less effective. Up to 4 and 6 kJ/m2 of 365-nm radiation, MePyPs and PyPs, respectively, were less mutagenic than 8-MOP but became more mutagenic at higher doses. At equal survival, the pyridopsoralens were, however, clearly less mutagenic than the bifunctional furocoumarins 8-MOP and 5-MOP. By plotting the genetic data versus the number of lesions induced in DNA, it was shown that the monoadducts induced by the monofunctional furocoumarins MePyPs and 3-CPs exert a relatively low potential for the induction of lethal and nuclear genetic events as compared to photoadditions induced by the bifunctional furocoumarins 8-MOP and 5-MOP. However, at a very high density, the monoadducts induced by MePyPs became as lethal and as mutagenic as the mixture of mono- and biadducts induced by 8-MOP and 5-MOP probably due to overloading of cellular repair capacities.(ABSTRACT TRUNCATED AT 400 WORDS)

DECREASED PHOTO-INDUCED MUTAGENICITY OF MONO-FUNCTIONAL AS OPPOSED TO BI-FUNCTIONAL FUROCOUMARINS IN YEAST

Abstract— The photoaddition of mono‐functional furocoumarins such as 5,7‐dimethoxycoumarin, angelicin and 3‐carbethoxypsoralen induces as a function of dose or of survival a much lower frequency of reverse mutations in Saccharomyces cerevisiae than the two bi‐functional furocoumarins, psoralen and 8‐methoxypsoralen. From this, it is suggested that bi‐functional lesions, i.e. DNA interstrand cross‐links, may be repaired in yeast by an error‐prone process.

Photochemotherapy using pyridopsoralens

Aiming to decrease the acute side effects and genotoxic hazards of PUVA, pyrido (3,4-C) psoralen (PP) and 7-methyl pyrido (3,4-C) psoralen (MPP) were synthesized and studied. Their UVA maximum absorption lies at 325 and 330 nm, respectively. Their photostability is comparable to that of 8-MOP. They complex to DNA in the dark, and, in the presence of UVA, produce only monoadditions to DNA, as shown by fluorescence and DNA denaturation-renaturation studies. In diploid eukaryotic yeast they are more effective than 8-MOP for the induction of lethal effects and mitochondrial damage. Their mutagenic activity per unit dose of UVA is in the same range as that of 8-MOP. However, per viable cell they are clearly less mutagenic than 8-MOP. This difference is also observed for recombinogenic activity. No oxygen effect is observed. In mammalian cells the following ranges of effectiveness are found: inhibition of DNA synthesis in human fibroblasts: MPP greater than PP greater than 8-MOP; mutagenic activity in V79 Chinese hamster cells: MPP greater than PP greater than 8-MOP; cell transforming ability in C3H embryonic mouse cells: MPP greater than 8-MOP greater than PP as a function of UVA dose, and: 8-MOP greater than MPP greater than PP as a function of survival; induction of sister chromatic exchanges (SCE) per unit dose: MPP greater than PP greater than 8-MOP in the linear part of the induction curve, and : 8-MOP greater than PP greater than MPP at the maximum level of SCE obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Some properties of the triplet excited-state of the photosensitizing furocoumarin - 3-carbethoxypsoralen.

Abstract— 3‐Carbethoxypsoralen (3‐CPs) has been tested in the photochemotherapy of psoriasis. It only forms monoadducts with DNA and is being considered as a non‐carcinogenic alternative to 8‐MOP which itself forms DNA crosslinks that arc difficult to repair. Using laser flash photolysis or pulse radiolysis, the triplet state of 3‐CPs, a possible intermediate in photosensitization, has been generated in several solvents: ethanol, water and benzene. The triplet lifetime, extinction coefficient and quantum efficiency of formation have been measured. Triplet reactivities towards (i) the solvents used, (ii) 3‐CPs, (iii) oxygen, (iv) tryptophan and (v) tyrosine, leading, respectively, to photoadditions with water, ethanol and 3‐CPs, to 1O2, semioxidized tryptophan and semioxidized tyrosine, (vi) thymine and (vii) uracil have been investigated. The dark binding of 3‐CPs to DNA has been studied by comparing the reactivity of eaq‐ with free 3‐CPs, free DNA and the 3‐CPs DNA complex. Some photophysical and photochemical properties of 4′,5′di‐hydro‐3‐carbethoxypsoralen(DH–3‐CPs), model of the main fluorescent photo‐product of 3‐CPs, have also been investigated. Biological consequences of the photochemical properties of 3‐CPs andDH–3‐CPs have been studied in a cellular system (haploid yeast).

The fate of 8-methoxypsoralen-photoinduced DNA interstrand crosslinks in Fanconi's anemia cells of defined genetic complementation groups

The fate of 8-methoxypsoralen (8-MOP)-photoinduced DNA interstrand crosslinks was followed by alkaline elution in Fanconis anemia (FA) fibroblasts belonging to complementation groups A (FA 150 and FA 402) and B (FA 145) in comparison to a normal (1 BR/3) and a heterozygote (F 311) cell line. Clonogenic cell survival to 8-MOP photoaddition was established in parallel for all cell lines. In comparison to normal cells, group A FA cells demonstrated a higher photosensitivity than group B cells (sensitivity index 2.3 and 1.5, respectively), the heterozygote cell line being only slightly more sensitive. FA cells from both groups A and B demonstrated an incision capacity of crosslinks, the kinetics and extent of which being, however, different from that of normal or heterozygote cells. The incision is slower in FA cells and, at 24 h of post-treatment incubation, the amount of crosslinks incised is clearly lower than that observed in normal cells for group A cells, whereas in group B cells incision approaches the level of normal cells. These results correlate with survival as well as with rates of DNA semi-conservative synthesis after 8-MOP photoaddition.