Abnormal rearrangements associated with V(D)J recombination in fanconi anemia¹

Abstract

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.

Introduction

Fanconi anemia (FA) belongs to a group of rare inherited autosomal recessive disorders in which anomalies in the processing of specific DNA lesions and chromosomal instability are associated with a predisposition to cancer. FA is characterised clinically by progressive bone marrow failure, developmental abnormalities and an increased risk of developing acute myelogenous leukemia (Auerbach et al., 1989). The hallmark of the FA phenotype is a high level of chromosomal breakage that is amplified after treatment with DNA cross-linking Auerbach and Allen 1991, Liu et al 1994. Eight FA genetic complementation groups (A to H) have been defined by somatic cell hybridisation studies Joenje et al 1997, Strathdee et al 1992a. The cDNAs for FA-C (Strathdee et al., 1992b) and FA-A have been cloned The Fanconi anemia/Breast cancer consortium 1996, Lo Ten Foe et al 1996 and mapped to chromosomes 9q22 and 16q24.3, respectively. The gene belonging to complementation group D has been assigned to the short arm of chromosome 3 (Whitney et al., 1995). Analysis of the FAA and FAC genes and their predicted amino acid sequences has revealed no significant homologies to other known genes.

The increased genomic instability in FA is reflected at the endogenous HPRT locus by a high proportion of deletions (Papadopoulo et al., 1990a). Knowing that misrepaired double-strand breaks (DSB) may lead to chromosomal rearrangements, the question arose as to whether anomalies in the processing of DSB may account for the excessive production of deletions in FA. To answer this question, we recently analysed the fate of a restriction endonuclease-introduced DSB within an extrachromosomal substrate. The data from these experiments indicate that mutations in FA genes lead to a marked decrease in the fidelity of non-homologous end-joining (NHEJ), as evidenced by a higher deletion frequency and an increased size of deletions Escarceller et al 1997, Escarceller et al 1998.

In mammalian cells, NHEJ and homologous recombination represent two major pathways for the repair of a DSB. DSB are induced by ionising radiation and radiomimetic chemicals and also arise during cellular metabolism (reactive oxygen species, replication, intermediate steps during the repair of some lesions, i.e. DNA interstrand cross-links). Moreover, the introduction of DSB plays a crucial role in important programmed biological processes, such as meiotic recombination, mating type switching in yeast and site-specific V(D)J recombination in vertebrates.

V(D)J recombination is the mechanism by which the genes encoding immunoglobulin (Ig) and T-cell-receptors (TCR) are assembled through the fusion of two or more gene segments, termed V, D and J, in developing B and T lymphocytes (for a review see Gellert 1996, Lieber 1991, Zhu and Roth 1996). The V(D)J reaction is initiated by a DSB at the recombination signal sequences (RSS), which are adjacent to the V, D and J segments Agrawal and Schatz 1997, van Gent et al 1995. It has been well documented that the introduction of a DSB requires the expression of the lymphoid-specific recombination activating genes, RAG1 and RAG2Oettinger et al 1990, Schatz et al 1989. The expression of these genes in any cell type is sufficient to initiate the V(D)J process in reporter recombination substrates (Oettinger et al., 1990).

To determine how a DSB, mediated by the expression of the RAG1 and RAG2 genes, is processed in FA cells, we set up a V(D)J recombination assay. Two extrachromosomal substrates, pHRecCJ and pHRecSJ, allowed us to examine V(D)J rearrangements with coding and signal joints as well as to detect unintended end-joining events produced in the region around the RSS. The molecular analysis of recombinants shows that, while the RAG1 and RAG2 initiated V(D)J recombination proceeds with high precision in normal human lymphoblasts, in FA-C and FA-D cells a significant proportion of rearrangements are abnormal, especially for coding joint formation.