Hypomutability in Fanconi anemia cells is associated with increased deletion frequency at the HPRT locus.

Abstract

Fanconi anemia (FA) is an inherited human disorder associated with a predisposition to cancer and characterized by anomalies in the processing ofDNA cross-links and certain monoadducts. We reported previously that the frequency of psoralen-photoinduced mutations at the HPRT locus is lower in FA cells than in normal cells. This hypomutability is shown here to be associated with an increased frequency of deletions in the HPRT gene when either a mixture ofcross-links and monoadducts or monoadducts alone are induced. Molecular analysis of mutants in the HPRT gene was carried out. In normal cells the majority of spontaneous and induced mutants are point mutations whereas in FA deletion mutations predominate. In that case a majority of mutants were found to lack individual exons or small clusters of exons whereas in normal cells large (complete or major gene loss) and small deletions are almost equally represented. Thus we propose that the FA defect lies in a mutagenic pathway that, in normal cells, involves bypassing lesions and subsequent gap flling by a recombinational process during replication.Cells possess various mechanisms to repair DNA lesions in order to maintain cell viability and the functional stability of DNA. Lesions unrepaired or repaired inaccurately lead to changes in the genetic information. These heritable changes range from base substitutions to large chromosomal rearrangements. Knowledge of the DNA lesions produced by a mutagen, coupled with an analysis of the molecular nature of the induced mutations, can give clues as to which lesions are biologically relevant and how these lesions are processed. In addition, the determination of mutagenic specificity in the presence or absence of a particular repair system can suggest mechanisms by which this system modulates the mutagenic process. Mutational changes are implicated in human health problems, including genetic diseases and cancer. Thus it is important to acquire information directly from human cells. Molecular techniques have made it possible to examine the molecular events underlying mutations at specific loci in mammalian cells (for reviews, see refs. 1 and 2). The following systems have been established for this purpose: shuttle vectors (for review, see ref. 3 and references therein), single integrated copies of a transfected bacterial gene (4), or analysis of mutations at endogenous loci (5-7). Among these systems, the last is the most laborious, but the advantage is that the chromosomal location, the structural conformation of DNA, and the topological constraints correspond to a natural situation. Among the various endogenous genes studied, the HPRT gene has several advantages: its large size (45 kilobases), its X chromosome location, and the availability of hybridization probes and of nucloeotide sequence data (8, 9). Moreover, large deletions or rearrangements can be detected since the HPRT locus is not close to essential genes (6, 10).Cells from patients with defects in DNA-damage processing provide opportunities to explore the m...

Biallelic inactivation of REV7 is associated with Fanconi anemia

“…In this role, REV7 acts as an adaptor protein between REV1 and REV3, the catalytic subunit of the POL ζ complex. A deficiency in REV7 could account for the well-known hypomutability of FA cells, resulting from a defect in TLS repair (20)(21)(22)(23). Second, REV7 plays a role in mitosis by preventing premature activation of the ure 3, C-F).…”

Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair

“…Although the recombinogenic DT40 B cells may magnify repair defects that are substantially milder in mammalian cells, it should be noted that fancc mutant DT40 cells also show only a mild HDR defect (20). Nevertheless, the mild impairment could certainly be causative for the increased frequencies of chromosome aberrations, spontaneous HPRT deletions (32), and loss of heterozygosity (33) observed in FA cells, as well as for the tumor predisposition of patients (14,15). Mice with weak hypomorphic alleles of Brca1 (8) and Brca2 (9) have only small reductions in HDR (i.e., 5-fold), yet they manifest chromosomal instability, developmental defects, and cancer predispositon (34)(35)(36).…”

Remodeling of DNA replication structures by the branch point translocase FANCM

“…First, deletions represent the most prevalent class of spontaneous mutations in FA cells, whereas base substitutions usually predominate in cells derived from healthy donors (34,35). Second, the knockdown of fancg in hamster CHO cells leads to an increase in the ratio of spontaneous deletions versus base substitutions (36).…”