Induction of recombination between diverged sequences in a mammalian genome by a double-strand break

Abstract

To investigate whether mammalian cells can carry out recombinational double-strand break (DSB) repair between highly diverged sequences, mouse fibroblasts were transfected with DNA substrates that contained a "recipient" thymidine kinase (tk) gene disrupted by the recognition site for endonuclease I-SceI. Substrates also contained a linked "donor" tk gene sequence. Following DSB induction by I-SceI, selection for tk-expressing clones allowed recovery of repair events occurring by nonhomologous end-joining or recombination with the donor sequence. Although recombinational repair was most efficient when donor and recipient shared near-perfect homology, we recovered recombination events between recipient and donor sequences displaying 20 % nucleotide mismatch. Recombination between such imperfectly matched ("homeologous") sequences occurred at a frequency of 1.7 × 10(-7) events per cell and constituted 3 % of the DSB repair events recovered with the pair of homeologous sequences. Additional experiments were done with a substrate containing a donor sequence comprised of a region sharing high homology with the recipient and an adjacent region homeologous to the recipient. Recombinational DSB repair tracts initiating within high homology propagated into homeology in 11 of 112 repair events. These collective results contrasted with our earlier work in which spontaneous recombination (not intentionally induced by a DSB) between homeologous sequences occurred at an undetectable frequency of less than 10(-9) events per cell, and in which events initiating within high homology propagated into adjoining homeology in one of 81 events examined. Our current work suggests that homology requirements for recombination are effectively relaxed in proximity to a DSB in a mammalian genome.

Mismatch tolerance during homologous recombination in mammalian cells

“…When DNA recombination happens between imperfectly matching sequences (synonymous with diverged sequences and homeologous sequences), the restored DNA molecule would have certain DNA sequence replaced by that of the DNA template. These recombination events with a homeologous conversion tract are thus called homeologous recombination events (HeR events) (53)(54)(55)(56). Due to HeR's deleterious consequences to gene function and genome stability, a lot of research have been done to address the mechanism how cells avoid DNA recombination between diverged sequences.…”

“…Pursuing the previous findings in our laboratory (53,55), a systematic research has been done to explore the mechanism how mammalian cells exclude highly diverged sequences from DNA recombination, and the results are presented in current dissertation.…”

Intrachromosomal recombination between highly diverged DNA sequences is enabled in human cells deficient in Bloom helicase

“…AW91 is composed of 20 nucleotides from the noncoding strand of the neomycin gene mapping 25 through 44 bp downstream from the neomycin start codon, with an Sp6 primer appended to the 5' end of the primer. PCR was carried out using Ready-To-Go PCR beads (GE Healthcare) and a "touchdown" PCR protocol as previously described (17). PCR products were expected to be ∼1.4 kb in length unless detectable deletions or insertions had occurred.…”

“…We also reported that when a spontaneous recombination event occurs within highly homologous sequences, mammalian cells fastidiously exclude adjoining mismatched bases from gene conversion tracts (15). Recently, we observed that the exclusion of mismatches from gene conversion events initiated within high homology is somewhat relaxed when the mismatches are positioned close to the DSB initiating the HR event (17). Nonetheless, the need for a stretch of high homology for the initiation of HR remains intact in proximity to a DSB (17).…”

“…Recently, we observed that the exclusion of mismatches from gene conversion events initiated within high homology is somewhat relaxed when the mismatches are positioned close to the DSB initiating the HR event (17). Nonetheless, the need for a stretch of high homology for the initiation of HR remains intact in proximity to a DSB (17).…”