Interhomolog recombination and loss of heterozygosity in wild-type and Bloom syndrome helicase (BLM)-deficient mammalian cells

Abstract

Genomic integrity often is compromised in tumor cells, as illustrated by genetic alterations leading to loss of heterozygosity (LOH). One mechanism of LOH is mitotic crossover recombination between homologous chromosomes, potentially initiated by a double-strand break (DSB). To examine LOH associated with DSBinduced interhomolog recombination, we analyzed recombination events using a reporter in mouse embryonic stem cells derived from F1 hybrid embryos. In this study, we were able to identify LOH events although they occur only rarely in wild-type cells (≤2.5%). The low frequency of LOH during interhomolog recombination suggests that crossing over is rare in wild-type cells. Candidate factors that may suppress crossovers include the RecQ helicase deficient in Bloom syndrome cells (BLM), which is part of a complex that dissolves recombination intermediates. We analyzed interhomolog recombination in BLM-deficient cells and found that, although interhomolog recombination is slightly decreased in the absence of BLM, LOH is increased by fivefold or more, implying significantly increased interhomolog crossing over. These events frequently are associated with a second homologous recombination event, which may be related to the mitotic bivalent structure and/or the cell-cycle stage at which the initiating DSB occurs.double-strand break repair | gene conversion L oss of heterozygosity (LOH) is a major mechanism for uncovering mutations in tumor-suppressor genes. Examination of tumor cells and model studies in cell lines have established several mechanisms of LOH, including chromosome loss/duplication (uniparental disomy), mitotic recombination between homologous chromosomes, and gross chromosomal rearrangement as a result of genome-wide chromosomal instability (1-4). LOH caused by homologous recombination (HR) can arise from crossing over between homologous chromosomes, followed by segregation of crossover (CO) chromatids to separate daughter cells (CO-LOH). In this way, the entire chromosome arm distal to the point of crossing over is homozygozed for genetic information. This process contrasts with interhomolog noncrossover (NCO) events during which gene conversion gives rise to only small regions of LOH (5). Chromosomal double-strand breaks (DSBs) may be initiating lesions for CO-LOH, because they induce interhomolog crossing over in meiotic cells (reviewed in ref. 6). Although DSB-induced crossing over is clearly necessary during the specialized first meiotic division (6), a role for interhomolog crossing over in mitotic cells is uncertain.DSBs induce mitotic interhomolog HR in mammalian cells (7), but CO-LOH occurs infrequently during DSB repair (5, 8), suggesting that factors actively suppress crossing over. Candidate factors include the RecQ helicase deficient in cells from Bloom syndrome patients (BLM) (9). Consistent with this notion, levels of spontaneous intersister COs, i.e., sister-chromatid exchanges, are elevated in both human and mouse BLM-deficient cells (10, 11), as is the frequency of spontaneous LOH (9...

Enhancement of microhomology-mediated genomic rearrangements by transient loss of mouse Bloom syndrome helicase

“…S5), while those of the other six clones (I-14, -16, -17, -63, -77, and G-10) remained either intact (I-14, -16, -63, and G-10) or slightly modified (I-17 and -77) on the B6 allele (data not shown). The recovery of the I-SceI allele to the wild type can be explained by interhomolog NCO recombination in Blm-deficient ES cells, as explained in a previous paper (Larocque et al 2011), and relatively short extents of gene conversion were observed (Supplemental Fig. S5, brackets).…”

“…The number of Dox(+)ISceIo(+) and Dox(+) ISceIo(-) colonies was significantly higher than that of Dox(-)ISceIo(+) and Dox(-)ISceIo(-) colonies, respectively, demonstrating that transient loss of BLM elevated the rate of LOH both in DSB-induced and uninduced conditions (Fig. 1F) (Larocque et al 2011). The occurrence of somatic LOH could be dependent on the cell-cycle phase.…”

“…A previous study demonstrated that site-directed DNA double-strand break (DSB) induction enhanced the rate of CO-mediated LOH in Blm-deficient (Blm tet/tet ) ES cells (Larocque et al 2011), suggesting that the DSB is a component of the mechanism of somatic LOH in Bloom syndrome patients. To provide a comprehensive view of the early processes of LOH in the absence of BLM, here, we present a molecular characterization of the genomic Schematic representation of the Dox-inducible knockdown construct for the Blm tet/tet ES cell line .…”

Essential Functions of C Terminus of Drosophila Topoisomerase IIIα in Double Holliday Junction Dissolution

“…The complex of Bloom helicase (Blm) and topoisomerase III␣ (Top3␣) 2 has been shown to convergently migrate double Holliday junctions (dHJs), an intermediate of double-strand break repair, into solely non-crossover products (1,2). The significance of this anti-recombinogenic function can be seen in the phenotype of Blm-deficient cells, which show a marked increase in sister chromatid exchanges (3) and loss of heterozygosity (4).…”

Replication stress and cancer: It takes two to tango

“…Their activity is restricted to the G2/M phases of the cell cycle where they are in charge of the resolution of persistent Holliday junctions that have not been dissolved by the helicase system (Figure 1). The resolution by endonucleases implies the generation of double strand breaks and favours their repair through non-homologous end joining, an error prone pathway that may increase genome instability [70]. Interestingly, a recent report indicates that the inactivation of these endonucleases also reduces replication fork speed, highlighting their important mechanism as a back-up repair system [71].…”