Dora Papadopoulo

A single mutated BRCA1 allele leads to impaired fidelity of double strand break end-joining

Heterozygosity for mutations in the BRCA1 gene in humans confers high risk for developing breast cancer, but a biochemical basis for this phenotype has not yet been determined. Evidence has accumulated implicating BRCA1, in the maintenance of genomic integrity and the protection of cells against DNA double strand breaks (DSB). Here we present evidence that human cells heterozygous for BRCA1 mutations exhibit impaired DNA end-joining, which is the major DSB repair pathway in mammalian somatic cells. Using an in vivo host cell end-joining assay, we observed that the fidelity of DNA end-joining is strongly reduced in three BRCA1+/− cell lines in comparison to two control cell lines. Moreover, cell-free BRCA1+/− extracts are unable to promote accurate DNA end-joining in an in vitro reaction. The steady-state level of the wild type BRCA1 protein was significantly lower than the 50% expected in BRCA1+/− cells and thus may underlie the observed end-joining defect. Together, these data strongly suggest that BRCA1 is necessary for faithful rejoining of broken DNA ends and that a single mutated BRCA1 allele is sufficient to impair this process. This defect will compromise genomic stability in BRCA1 germ-line mutation carriers, triggering the genetic changes necessary for the initiation of neoplastic transformation.

Increased radiosensitivity of granulocyte macrophage colony-forming units and skin fibroblasts in human autosomal recessive severe combined immunodeficiency.

We studied the radiosensitivity of granulocyte macrophage colony-forming units (GM-CFU) in patients with a severe combined immunodeficiency (SCID). Three patients lacking both mature T and B cells showed a twofold higher GM-CFU radiosensitivity calculated as the DO value (dose required to reduce survival to 37%), and an identical observation was made with fibroblasts from one of these patients. A patient with an SCID with hypereosinophilia, i.e., Omenns syndrome characterized by extremely restricted T cell heterogeneity and a lack of B cells, also showed abnormal GM-CFU radiosensitivity. In contrast, GM-CFU from a patient lacking only T cells (X-linked form of SCID) showed normal GM-CFU radiosensitivity. These data further support the similarity between human T(-) B(-) SCID and the murine acid mutation characterized by a defect in T cell receptor and immunoglobulin gene rearrangement, and by an abnormal double-strand DNA break repair function. In addition, they strongly suggest that the Omenns immunodeficiency syndrome may be a leaky T(-)B(-) SCID phenotype as previously indicated by the coexistence of the two phenotypes in siblings.

Two complementation groups of Fanconi's anemia differ in their phenotypic response to a DNA-crosslinking treatment

SummaryThe two genetic complementation groups reported for Fanconis anemia (FA) correspond to two phenotypic classes as characterized by measurements of the rate of DNA semiconservative synthesis after 8-methoxypsoralen photo-addition. This test allows a rapid genetic classification of FA patients which appears to be a prerequisite for investigations of the biochemical defect(s) in FA.

The Transcriptional Histone Acetyltransferase Cofactor TRRAP Associates with the MRN Repair Complex and Plays a Role in DNA Double-Strand Break Repair

ABSTRACT Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.

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.

The fidelity of double strand breaks processing is impaired in complementation groups B and D of fanconi anemia, a genetic instability syndrome

In mammalian cells, nonhomologous end-joining is the predominant mechanism to eliminate DNA double strand breaks. Such events are at the origin of deletion mutagenesis and chromosomal rearrangements. The hallmark of Fanconi anemia, an inherited cancer prone disorder, is increased chromosomal breakage associated to over-production of deletions. Knowing that double strand breaks are at the origin of deletion mutagenesis, the question arises whether their processing is affected in FA. We set up a «host cell end-joining assay» to analyze the fate of double strand breaks into extrachromosomal substrates transiently replicated in normal and FA-D lymphoblasts. Although no difference in plasmid survival was found, blunt-ended breaks were sealed with significantly lower fidelity in FA cells, resulting in a higher deletion frequency and a larger deletion size. The results suggest that FA-D and FA-B gene products are likely to play a role in end-joining fidelity of specific DNA double strand breaks.

Frequencies of HPRT- lymphocytes and glycophorin A variants erythrocytes in Fanconi anemia patients, their parents and control donors

The mutant frequency of 6-thioguanine resistance (HPRT locus) in circulating T lymphocytes from 23 Fanconi anemia (FA) patients has been determined. The glycophorin A (GPA) in vivo cell mutants assay, which detects allele loss variant phenotypes arising from mutations in erythroid progenitor cells of GPA heterozygous MN individuals, has been applied in parallel to FA patients. No significant difference in frequency of HPRT- mutants was observed in FA compared to age matched healthy donors. In contrast, the mean frequency of GPA variant cells was elevated 31-fold for hemizygous NO variants and 8-fold for homozygous NN variants in FA patients over normal controls. In heterozygous FA parents, HPRT- mutant frequencies and GPA variant frequencies were within the normal range. Molecular analysis of HPRT- mutants has previously shown that FA cells have a high tendency to form deletions. Knowing that the cellular events allowing the detection of mutations at the HPRT and the GPA locus differ, our results emphasize the possible correlation between events of spontaneous loss of heterozygosity and genetic predisposition to cancer as observed in FA.

Fidelity of DNA double-strand break repair in heterozygous cell lines harbouring BRCA1 missense mutations.

Germ-line mutations of the BRCA1 and BRCA2 genes, when they lead to a truncated protein, confer a high risk of breast and ovarian cancer. However, the role of BRCA1 missense mutations in cancer predisposition is unclear. Functional assays may be very helpful to more clearly define the biological effect of these mutations, and could therefore be useful in clinical practice. A recent study using a Host Cell End-Joining assay showed that a truncating mutation results in impaired fidelity of DSB repair by DNA end-joining. In the present study, we examined the fidelity of DSB repair in four lymphoblastoid cell lines with BRCA1 missense mutations. The fidelity of DNA end-joining was impaired in the four cell lines studied compared to the normal control cell line. The fidelity of end-joining was similar to that of a truncated mutation control cell line for one cell line and slightly higher for the other cell lines.

Mutagenic effects photoinduced in mammalian cells in vitro by two monofunctional pyridopsoralens

Abstract— The photobiological activity of the two monofunctional pyridopsoralens pyrido (3,4‐c) psoralen (PyPs) and 7‐methyl pyrido (3,4‐c) psoralen (MePyPs) was studied in mammalian cells in vitro taking 8‐methoxypsoralen (8‐MOP) as a reference compound.

HIGH LEVELS OF 4,5′,8-TRIMETHYLPSORALEN PHOTOINDUCED FURAN-SIDE MONOADDUCTS CAN BLOCK CROSS-LINK REMOVAL IN NORMAL HUMAN CELLS

Abstract— The induction and removal of DNA interstrand cross‐links (CL) was studied in normal human fibroblasts (1BR/3) using the highly photoreactive furocoumarin 4,5′,8‐trimethylpsoralen (TMP) in combination with monochromatic 365 nm and/or 405 nm radiation. We report that the presence of large amount of furan‐side monoadducts (MAf) induced by TMP plus 365 nm radiation blocks CL incision. When the amount of MAf is reduced by their conversion into even more CL, incision of the cross‐links is more efficient.