Loss of heterozygosity induced by a chromosomal double-strand break

Abstract

The repair of chromosomal double-strand breaks (DSBs) is necessary for genomic integrity in all organisms. Genetic consequences of misrepair include chromosomal loss, deletion, and duplication resulting in loss of heterozygosity (LOH), a common finding in human solid tumors. Although work with radiation-sensitive cell lines suggests that mammalian cells primarily rejoin DSBs by nonhomologous mechanisms, alternative mechanisms that are implicated in chromosomal LOH, such as allelic recombination, may also occur. We have examined chromosomal DSB repair between homologs in a gene targeted mammalian cell line at the retinoblastoma (Rb) locus. We have found that allelic recombinational repair occurs in mammalian cells and is increased at least two orders of magnitude by the induction of a chromosomal DSB. One consequence of allelic recombination is LOH at the Rb locus. Some of the repair events also resulted in other types of genetic instability, including deletions and duplications. We speculate that mammalian cells may have developed efficient nonhomologous DSB repair processes to bypass allelic recombination and the potential for reduction to homozygosity.

Figure 1

Gene-targeted region of the Rb locus in ES cells containing recombination substrates. (A) The Rb locus at exons 19 and 20. (B) Gene-targeted Rb alleles containing substrates to detect DSB-induced allelic recombination. Alleles K and L are nondisrupting Rb alleles that have the recombination substrates gene targeted to intron 18. Alleles M and N are disrupting Rb alleles that have the recombination substrates targeted to exon 19. Each Rb allele contains a defective neo gene. The K and M alleles contain the Sneo gene that is mutated by the presence of an I-SceI site at the 3′ end of the neo gene. The 18-bp I-SceI site can be cleaved in vivo by expression of I-SceI. The L and N alleles contain the Pneo gene that is mutated at its 5′ end by the insertion of a PacI linker. Transcription of the neo genes is opposite to that of the Rb gene. Drug selection markers hyg⁺ and hprt⁺ are transcribed in the same orientation as the Rb gene and were used to select for gene targeting in the hprt⁻ ES cell line. Single rounds of gene targeting were used to construct cell lines with each of the K, L, M, and N alleles. Consecutive rounds of gene targeting were used to construct cell lines with KL and MN alleles. The KL cell line is effectively Rb^+/+, whereas the MN cell line is Rb^−/−.

Figure 2

Southern blot analysis of G418^R subclones obtained after transfection of cell lines with the I-SceI expression vector. (A) Structure of Rb alleles with relevant restriction sites. Parentheses indicate absence of exon 19 in the M and N alleles. HindIII–PacI and PstI restriction fragment sizes in kb are indicated below each allele. Neo⁺ clones derived from DNA end-joining or allelic recombination with gene conversion tracts of less than 300 bp (i.e., STGC) give indistinguishable products in which the only detectable alteration of the K and M alleles is loss of the I-SceI site from the neo gene, as indicated. Recombinant hybrid alleles are derived from allelic recombination with gene conversions tracts that are greater than 300 bp (i.e., LTGC). LTGCs that include the entire hprt gene are at least 2.8 kb. In addition to the neo⁺ hybrid allele, each recombinant cell line has an intact L or N allele, as shown. (B) Southern blot analysis of genomic DNA cleaved with HindIII/PacI. (C) Southern blot analysis of genomic DNA cleaved with PstI. Genomic DNA of expanded K, M, KL, and MN G418^R clones was digested with restriction enzymes as noted and hybridized to a ³²P-labeled neo EagI–NcoI fragment. Rb alleles in these clones have fragment sizes as shown in A, except for KL neo⁺ clone C12. The hybrid Rb allele in clone C12 has an approximate 2.5-kb deletion. This is detected by the faster mobility of the upper band in B and is indicated by the arrow in C.

Figure 3

Southern blot analysis demonstrating loss of the I-SceI site and duplication of the Rb locus. Lanes are marked with W (for wild-type) if alleles are unaltered in copy number and D if an allele is duplicated. The neo gene probe is as in Fig. 2. (A) Analysis of genomic DNA cleaved with BglII/I-SceI from KL neo⁺ clones. Restriction fragment sizes are indicated for each of the alleles, with both the parental and neo⁺ alleles shown for K (K-Sneo and K-neo+, respectively). The KL clone in lane De has a deletion in the recombinant L–K allele (clone C6; see text). (B) Analysis of genomic DNA cleaved with BglII/I-SceI from MN neo⁺ clones. Restriction fragment sizes are indicated for each of the alleles, with both the parental and neo⁺ allele shown for M (M-Sneo and M-neo+, respectively). All MN clones in lanes labeled D have duplicated N alleles, except one clone that has an additional parental M-Sneo allele, as indicated.