Fine-Resolution Analysis of Products of Intrachromosomal Homeologous Recombination in Mammalian Cells

Abstract

Mouse Ltk؊ cell lines that contained a herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene with a 16-bp insertion mutation linked to either a defective HSV-2 tk gene or a hybrid tk sequence comprised of HSV-1 and HSV-2 tk sequences were constructed. HSV-1 and HSV-2 tk genes have 81% nucleotide identity and hence are homeologous. Correction of the insertion mutant HSV-1 tk gene via recombination with the hybrid tk sequence required an exchange between homeologous tk sequences, although recombination could initiate within a region of significant sequence identity. Seven cell lines containing linked HSV-1 and HSV-1-HSV-2 hybrid tk sequences gave rise to tk ؉ segregants at an average rate of 10 ؊8 events per cell division. DNA sequencing revealed that each recombinant from these lines displayed an apparent gene conversion which involved an accurate transfer of an uninterrupted block of information between homeologous tk sequences. Conversion tract lengths ranged from 35 to >330 bp. In contrast, cell lines containing linked HSV-1 and HSV-2 tk sequences with no significant stretches of sequence identity had an overall rate of homeologous recombination of <10 ؊9. One such cell line produced homeologous recombinants at a rate of 10 ؊8. Strikingly, all homeologous recombinants from this latter cell line were due to crossovers between the HSV-1 and HSV-2 tk genes. Our results, which provide the first detailed analysis of homeologous recombination within a mammalian genome, suggest that rearrangements in mammalian genomes are regulated by the degree of sequence divergence located at the site of recombination initiation.Recombination between similar but imperfectly matched sequences is referred to as homeologous recombination. Homeologous recombination events have been recovered in several organisms, including yeast (2,12,15,16,18,31,32,34,35,40,43), bacteria (28, 36-39, 44, 45), and mammalian cells (3,53). When homeologous recombination is detected, it is typically much less frequent than is recombination between highly homologous sequences. Low rates for homeologous recombination may help to conserve genomic integrity by preventing undesirable rearrangements. On the other hand, when homeologous recombination does occur, it can potentially lead to novel genes and chromosomes and may play an important role in the evolution of genomes. For these reasons, it is of considerable interest to gain knowledge about such events as they occur in a variety of organisms. We previously investigated intrachromosomal homeologous recombination between a defective herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) sequence and a closely linked HSV-2 tk sequence in the genome of mouse fibroblasts (52). HSV-1 and HSV-2 tk sequences have 81% sequence identity, with a 35-bp stretch of sequence identity being the longest (19, 49). The rate of intrachromosomal homeologous recombination between HSV-1 and HSV-2 tk sequences was reduced more than 1,000-fold compared with that of intrachromosomal recombination between nearly perfect...

Genetic Exchange Between Homeologous Sequences in Mammalian Chromosomes Is Averted by Local Homology Requirements for Initiation and Resolution of Recombination

“…Strand exchange can penetrate homeologous sequences: In considering how the fastidious exclusion of homeologous sequence from recombination products may be brought about, we bore in mind our earlier studies in which substrates that contained hybrid donors (and were similar to pHYB12-8 and pHYB21A) yielded gene conversion tracts that in some cases included .300 bp of homeologous sequence (Yang and Waldman 1997). The exclusion of homeologous sequence from gene conversion tracts in our current work seemed at odds with those earlier studies.…”

“…Experiments with pHYB21A-28 in conjunction with our earlier studies (Yang and Waldman 1997;Waldman et al 1999) suggest that certain DNA motifs or sequences might influence recombination. In pHYB21A-28, the XhoI linker in the recipient tk gene is positioned opposite the homeologous portion of the donor when recipient and donor are paired (Figure 1).…”

“…In previous investigations we found not only that small degrees of sequence divergence can suppress intrachromosomal recombination but also that cells can potentially carry out accurate exchanges between homeologous sequences if homeologous sequences are located adjacent to a region of high homology (Waldman and Liskay 1988;Yang and Waldman 1997). Such findings previously led us to conclude that stringent homology requirements are involved almost exclusively in the initiation of recombination and, once initiated, a recombination event can propagate into sequences displaying a high degree of divergence.…”

Transcription of a Donor Enhances Its Use during Double-Strand Break-Induced Gene Conversion in Human Cells

“…However, the donor loci in this system were not identical, and the focus of the study was on the efficiency of the homology search and whether the search occurs near or far from broken ends. In mammalian cells, HR efficiency is reduced by heterologies (16,79), and this is likely to influence donor preference when potential donors differ in the degree of sequence similarity to a recipient locus. In mouse cells, Tremblay et al (70) showed that DSBs at an I-SceI site within a LINE element were repaired by conversion involving other LINE elements and that the most commonly used donors were those most active in retrotransposition.…”

Functionally Tolerable Mutations Incorporated in Proteins Guides the Evolution Process

“…While diversions exist for every evolved factor among different species, there are some factors which constantly catalyze and assist the associated evolutionary track. Such factors including the Mutation (be it lethal [9] or linked [10][11][12][13]), Genetic Recombination & count of such sites occurring during the crossing-overs [14][15][16][17] and Environmental Constraints form the primary tools to pave the path for evolution. Such factors normally act along with several other key constraints together to demarcate the evolutionary path.…”