Genetic exchange between homeologous sequences in mammalian chromosomes is averted by local homology requirements for initiation and resolution of recombination

Abstract

We examined the mechanism by which recombination between imperfectly matched sequences (homeologous recombination) is suppressed in mammalian chromosomes. DNA substrates were constructed, each containing a thymidine kinase (tk) gene disrupted by insertion of an XhoI linker and referred to as a "recipient" gene. Each substrate also contained one of several "donor" tk sequences that could potentially correct the recipient gene via recombination. Each donor sequence either was perfectly homologous to the recipient gene or contained homeologous sequence sharing only 80% identity with the recipient gene. Mouse Ltk(-) fibroblasts were stably transfected with the various substrates and tk(+) segregants produced via intrachromosomal recombination were recovered. We observed exclusion of homeologous sequence from gene conversion tracts when homeologous sequence was positioned adjacent to homologous sequence in the donor but not when homeologous sequence was surrounded by homology in the donor. Our results support a model in which homeologous recombination in mammalian chromosomes is suppressed by a nondestructive dismantling of mismatched heteroduplex DNA (hDNA) intermediates. We suggest that mammalian cells do not dismantle mismatched hDNA by responding to mismatches in hDNA per se but rather rejection of mismatched hDNA appears to be driven by a requirement for localized homology for resolution of recombination.

Figure 1.—

Recombination substrates. (Top) A schematic of a generic recombination substrate. The DNA construct is shown as if linearized at the unique ClaI site in the vector. Inserted between two BamHI sites (B) is a 2.5-kb fragment containing an HSV-1 tk gene disrupted by insertion of an XhoI (X) linker and referred to as a “recipient.” Inserted between two HindIII (H) sites is a truncated tk sequence referred to as a “donor.” The direction of transcription of recipient and donor tk sequences is from left to right, and the two tk sequences are separated by ∼4.4 kb. Below the generic substrate are schematics of the recipient and donor tk sequences contained in the six specific recombination substrates used. For each substrate, the recipient gene is shown on top with the donor gene aligned beneath it. Open rectangles represent HSV-1 tk sequences while stippled rectangles represent HSV-2 tk sequences. For pHYB21A-28 the recipient tk gene is mutant 28, while for all other substrates the recipient tk gene is mutant 8 (see materials and methods).

Figure 2.—

Slot blot analysis of recombination events recovered from cell lines containing pHYB12-8. Recipient tk gene sequence was PCR amplified from genomic DNA isolated from HAT^R segregants from cell lines containing pHYB12-8 and each PCR product was applied to two slot blots. One blot (I) was hybridized with probe 1, which is specific for HSV-1 tk sequence, while the other blot (II) was hybridized with probe 2, which is specific for HSV-2 tk sequence. Both probes mapped immediately downstream from the position of the junction between HSV-1 and HSV-2 tk sequences in the pHYB12-8 donor, as illustrated in III. (In III, open rectangles represent HSV-1 tk sequence and the stippled rectangle represents HSV-2 tk sequence.) The first row on each slot blot contains hybridization controls, with samples 1A and 1B containing HSV-1 tk sequence and sample 1C containing HSV-2 tk sequence. All other samples in rows 2–12 were derived from independent HAT^R segregants. In addition to the 33 HAT^R clones analyzed in the blots shown, an additional 11 HAT^R clones were analyzed in a similar fashion.

Figure 3.—

Nucleotide sequences of gene conversion tracts containing homeologous HSV-2 tk sequence. Nucleotide numbering is according to Wagner et al. (1981). The upper line of sequence is HSV-1 tk sequence from a portion of the recipient tk genes in pHYB21A and pHYB21A-28. The locations of the XhoI linker insertion in tk mutant 8 (the recipient gene in pHYB21A) and in tk mutant 28 (the recipient gene in pHYB21A-28) are indicated by labeled inverted triangles. The lower line of sequence is from a portion of the common hybrid donor tk sequence in both pHYB21A and pHYB21A-28, with the junction between HSV-2 and HSV-1 sequence shown. An asterisk is present at each position in the donor sequence where the donor is identical to the recipient HSV-1 tk sequence. In the HSV-2 tk portion of the donor, each nucleotide difference between donor HSV-2 tk and recipient HSV-1 tk sequence is indicated. Beneath the donor sequence is indicated the upstream-most HSV-2 tk marker in the gene conversion tracts of recovered clones. Clone K3-1 was recovered from cell line K3 containing pHYB21A. The gene conversion tract from clone K3-1 contains the HSV-2 “G” nucleotide indicated by the arrow as well as every downstream HSV-2 nucleotide marker through the position of the HSV-2/ HSV-1 junction. Clones 28-1–28-6 were recovered from lines containing HYB21A-28. The gene conversion tract from each clone contains the HSV-2 nucleotide indicated by the appropriate arrow as well as every downstream HSV-2 nucleotide marker through the position of the HSV-2/HSV-1 junction. Every gene conversion tract appeared to be continuous except for the tract from clone 28-5. Clone 28-5 displays a “C” (equal to HSV-1 sequence) at position 995 despite displaying 2 upstream and 25 downstream HSV-2 markers.