Transcription of a donor enhances its use during double-strand break-induced gene conversion in human cells.
Homologous recombination (HR) mediates accurate repair of double-strand breaks (DSBs) but carries the risk of large-scale genetic change, including loss of heterozygosity, deletions, inversions, and translocations. Nearly one-third of the human genome consists of repetitive sequences, and DSB repair by HR often requires choices among several homologous repair templates, including homologous chromosomes, sister chromatids, and linked or unlinked repeats. Donor preference during DSB-induced gene conversion was analyzed by using several HR substrates with three copies of neo targeted to a human chromosome. Repair of I-SceI nuclease-induced DSBs in one neo (the recipient) required a choice between two donor neo genes. When both donors were downstream, there was no significant bias for proximal or distal donors. When donors flanked the recipient, we observed a marked (85%) preference for the downstream donor. Reversing the HR substrate in the chromosome eliminated this preference, indicating that donor choice is influenced by factors extrinsic to the HR substrate. Prior indirect evidence suggested that transcription might increase donor use. We tested this question directly and found that increased transcription of a donor enhances its use during gene conversion. A preference for transcribed donors would minimize the use of nontranscribed (i.e., pseudogene) templates during repair and thus help maintain genome stability.
Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA. jnickoloff@salud.unm.edu
Ezra
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Schildkraut
Cheryl A
CA
Miller
Jac A
JA
Nickoloff
D000483:Alleles; D002460:Cell Line; D004249:DNA Damage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D005785:Gene Conversion; D017353:Gene Deletion; D006801:Humans; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014158:Transcription, Genetic
D029701:Saccharomyces cerevisiae Proteins; D003907:Dexamethasone; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific