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

Content Provider	Springer Nature Link
Author	Bhattacharjee, Vikram Lin, Yunfu Waldman, Barbara C. Waldman, Alan S.
Copyright Year	2013
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.
Starting Page	2359
Ending Page	2371
Page Count	13
File Format	PDF
ISSN	1420682X
Journal	Cellular and Molecular Life Sciences
Volume Number	71
Issue Number	12
e-ISSN	14209071
Language	English
Publisher	Springer Basel
Publisher Date	2013-11-21
Publisher Place	Basel
Access Restriction	Subscribed
Subject Keyword	Homeologous recombination Homologous recombination Double-strand break repair Mammalian cell culture Genomic stability Cell Biology Biomedicine general Life Sciences Biochemistry
Content Type	Text
Resource Type	Article
Subject	Cell Biology Molecular Biology Molecular Medicine Pharmacology Cellular and Molecular Neuroscience

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