Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability, hypersensitivity to DNA cross-linking agents, and a prolonged G2 phase of the cell cycle. We observed a marked dose-dependent accumulation of FA cells in the G2 compartment after treatment with 4,5′,8-trimethylpsoralen (Me3Pso) in combination with 365 nm irradiation. Using bivariate DNA distribution methodology, we determined the proportion of replicating and arresting S-phase cells and observed that, whereas normal cells arrested DNA replication in the presence of Me3Pso cross-links and monoadducts, FA lymphoblasts failed to arrest DNA synthesis. Taken together, the above data suggest that, in response to damage induced by DNA cross-linking agents, the S-phase checkpoint is inefficient in FA cells. This would lead to accumulation of secondary lesions, such as single- and double-strand breaks and gaps. The prolonged time in G2 phase seen in FA cells therefore exists in order to allow the cells to remove lesions which accumulated during the preceding abnormal S phase.

The principal cellular feature of Fanconi anemia (FA), an inherited cancer prone disorder, is a high level of chromosomal breakage, amplified after treatment with crosslinking agents. Three of the eight genes involved in FA have been cloned: FANCA, FANCC and FANCG. However, their biological functions remain unknown. We previously observed an excessive production of deletions at the HPRT locus in FA lymphoblasts belonging to the relatively rare complementation group D(1) and an increased frequency of glycophorin A (GPA) variants in erythrocytes derived from FA patients (2). In thi study, we examined the molecular nature of 31 HPRT mutations formed in vivo in circulating T-lymphocytes isolated from 9 FA male patients. The results show that in all FA patients investigated the deletions are by far the most prevalent mutational event in contrast to age matched healthy donors, in which point mutations predominate. The complementation group in the FA patients examined in the present study has not yet been defined. However, knowing that mutations in the FANCA and FANCC gene are found to be involved in at least 70% of the FA patients, it can be expected that the excessive production of deletions is a general feature of the FA phenotype. In addition, the spectrum of HPRT deletions observed in FA patients differs from that of healthy children: there is a high frequency of 3'-terminal deletions and a strikingly low proportion of V(D)J mediated events. Based on previous findings, a decreased fidelity of coding V(D)J joint formation (3) and an inaccurate repair of specific DNA double strand breaks via Non-Homologous End Joining (4), we propose that FA genes play a role in the control of the fidelity of rejoining of specific DNA ends. Such a defect may explain several basic features of FA, such as chromosomal instability and deletion pronenness.

The hallmark of Fanconi anemia (FA), a rare inherited cancer prone disorder, is a high level of chromosome breakage, spontaneous and induced by cross-linking agents. The increased genomic instability of FA is reflected at the gene level by an overproduction of intragenic deletions. Two of the eight FA genes have been cloned, however, their function remains unknown. We recently demonstrated that the lack of functional FA genes lead to a marked decrease in the fidelity of non-homologous end-joining, a pathway that mammalian cells predominantly use to repair DNA double-strand breaks (DSB). Knowing that specific DSB are generated during V(D)J recombination, here we have examined the molecular features of V(D)J rearrangements in normal and FA lymphoblasts belonging to complementation groups C and D. Using appropriate extrachromosomal recombination substrates, V(D)J coding and signal joint formation have been analysed quantitatively and qualitatively. Our results show that the frequency of coding and signal joint formation was not significantly different in normal and FA cells. However, when the fidelity of the V(D)J reaction was examined, we found that in normal human lymphoblasts V(D)J recombination proceeds with high precision, whereas, in FA cells a several fold increase in the frequency of aberrant rearrangements is associated with V(D)J coding joint formation. The abnormal recombinants that we recovered in FA are consistent with excessive degradation of DNA ends generated during the V(D)J reaction. On the basis of these findings, we propose a working model in which FA genes play a role in the control of the fidelity of rejoining of specific DNA ends. Such a defect may explain several basic features of FA, such as chromosomal instability and deletion proneness.

To assess the role of a given genotoxic agent in the etiology of human cancers, it is useful to establish the mutational specificity of this agent. The aim of this study was to investigate whether the processing of psoralen photolesions, interstrand cross-links (CL) and monoadducts (MA), leaves a specific molecular signature in the mutational events produced at an endogenous locus,HPRT. Human lymphoblasts were treated by 4,5′,8-trimethylpsoralen (Me3Pso) in association with a double irradiation protocol (365 plus 365 nm) which allows us to increase the proportion of CL for a given constant number of total photoadducts. The molecular spectrum of mutations at theHPRTlocus induced in these conditions was compared to the previously reported spectra of mutations induced by the same psoralen in combination with a single irradiation of either 365 nm (induction of MA and a low proportion of CL) or 405 nm (producing almost exclusively MA). In all treatment conditions, base substitutions constitute the major type of Me3Pso photoinduced mutations. The majority of base substitutions involve a T residue preferably within a 5′-TpA sequence which corresponds to the favoured sites of psoralen photoadducts. In other words, the Me3Pso photolesions induce at the endogenousHPRTlocus a highly specific signature. Moreover, base substitutions have been essentially found in the non-transcribed strand of the HPRT gene suggesting that the psoralen photolesions are preferentially removed from the transcribed strand. In spite of the considerable difference between the proportion of lesions of both types (CL or MA) induced in different treatment conditions, the kind of mutations and their sequence distribution are similar suggesting that the mutagenic processing of psoralen CL and MA is similar at least for the steps resulting in base substitutions.

Using the 4,5',8-trimethylpsoralen in combination with the reirradiation protocol, we show that, in normal human lymphoblasts, the cytotoxic potential of photoinduced cross-links (CL) is higher than that of monoadducts (MA). In contrast to cytotoxicity, the significant increase in the proportion of CL, at a constant level of total adducts, had no effect on the induction of mutations at the HPRT locus. Comparison with the data obtained in yeast and rodent cells using the same double irradiation protocol shows that the mutagenic potential of CL versus MA varies between species. This suggests that the equilibrium between the excision, the recombinational and the mutagenic components of the repair pathways which probably determine the mutagenic efficiency of CL versus MA is likely to be species-dependent.

Radioadaptation to the mutagenic effect of ionizing radiation by pre-exposure of human cells to a low dose has been shown to decrease the proportion of HPRT- mutants of the deletion type. To determine whether point mutations would be affected by the adaptive treatment, the molecular nature of mutations induced after exposure to low, high or low plus high doses was established. DNA sequencing of 38 point mutants which still expressed mRNA was performed using reverse transcription/polymerase chain reaction amplification. Under all conditions, base substitutions were the most common mutational event (range 72-80%), the remainder being frameshift and small deletions. The types and proportions of base changes did not appear to be differentially modified. A clustering of mutations was observed in exon 8, independently of the radiation protocol. About 40% of the mutants exhibited incorrect splicing of mRNA. The lack of striking modifications between the different molecular spectra of point mutations suggests that the low-dose pre-exposure does not affect the production and/or the processing of lesions leading to point mutations. Thus the highly significant effect triggered by the low dose is the preferential reduction of deletion-type mutations. In view of the actual small data set, definitive conclusions will be drawn only when our observations are confirmed or can be generalized to human endogenous loci other than the HPRT locus, which is particularly prone to the recovery of deletion-type mutations.

Spontaneous and induced chromosomal breakage is an important cellular feature of Fanconi anemia (FA), an inherited DNA repair disorder characterized by progressive bone marrow failure, developmental abnormalities, and predisposition to leukemia. We have previously reported that in comparison to normal cells, there is a substantial increase in frequency of intragenic deletions at an endogenous locus (HPRT) in FA lymphoblasts. Taken together with the increased chromosomal instability, these observations indicated that the wild-type FA gene(s) plays an important role in the maintenance of the genomic integrity. To obtain information on the mechanism(s) underlying the genomic rearrangements in FA, the breakpoint sites of deletions in 11 FA-derived HPRT- mutants were analyzed. The results indicate that a significant proportion of deletions involving a loss of a given exon are identical and that two deletions of different size have the same 3' breakpoint. Interestingly, it appears that in most of the mutants there is a common deletion signal sequence, which suggests that the mutations in the FA gene(s) may lead to an aberrant site-specific cleavage activity that might be responsible for the deletion proneness and the chromosomal instability characteristic of the FA pathology. From the similarity or even identity of the signal sequence at some of the breakpoints with the consensus heptamer which directs cleavage and joining in the assembly of immunoglobulin and T-cell receptor genes, we speculate that steps in common with the V(D)J recombinational process may be illegitimately involved in FA cells.

The molecular spectra of mutations photoinduced (405 nm) by 4,5',8-trimethylpsoralen monoadducts (MA), at an endogenous locus, hypoxanthine-guanine phosphoribosyl-transferase (HPRT) in normal and in a Fanconi anemia (FA) lymphoblast cell line, complementation group D, are presented. We show that, in normal cells, MA induce only base substitutions. In contrast, in FA cells which are partially deficient in the incision of MA, deletions are preferentially induced over point mutations (62% of the total). Although the proportion of base substitutions is lower in FA cells, their type and sequence distribution are similar in FA and normal cell lines. The majority of base substitutions are located at sites of psoralen MA which suggest that 4,5',8-trimethylpsoralen photoinduced mutations are targeted and preferentially formed in the non-transcribed strand. Moreover, point mutations induced by MA in normal and FA cells are not homogeneously distributed, they preferentially occur in exon 8 of the HPRT gene. This heterogeneous distribution of mutations is ascribed to processing of MA. Great similarities were found between normal and FA cells with respect to the nature and location of point mutation at the HPRT gene; the high proneness to deletions remains one of the major instability features of FA.

Molecular characterization of mutations photoinduced by a cross-linking agent, 4,5',8-trimethylpsoralen (Me3PSO), in normal human lymphoblasts was conducted in parallel with lymphoblasts derived from Fanconi anemia patients. Such cells have been previously described to be impaired in repair of psoralen photolesions. The endogenous HPRT locus was used as a target gene. The treatment of cells with Me3PSO in combination with 365 nm irradiation leads to the formation of interstrand cross-links, and specific monoadducts. Our analysis revealed that the mutagenic processing of Me3PSO photoadducts in normal human cells results essentially in base substitutions (84%). These are localized to sequences shown previously to be favored for the formation of Me3PSO monoadducts. The mutagenic processing of the same lesions in Fanconi anemia cells results in fewer base substitutions (22%), with deletions (66%) being the predominant class of mutation. In contrast to prokaryotic systems, frameshifts are poorly represented among Me3PSO induced mutations in human cells. In spite of important differences between the kinds of mutations observed in the two cell lines, our analysis reveals similarities in the type of base substitutions and their sequence distribution. In both normal and Fanconi anemia cell lines mutations, mostly targeted on thymine residues, are preferentially located on the non-transcribed strand.