Non-homologous end-joining for repairing I-SceI-induced DNA double strand breaks in human cells.
DNA double strand breaks (DSBs) are usually repaired through either non-homologous end-joining (NHEJ) or homologous recombination (HR). While HR is basically error-free repair, NHEJ is a mutagenic pathway that leads to deletion. NHEJ must be precisely regulated to maintain genomic integrity. To clarify the role of NHEJ, we investigated the genetic consequences of NHEJ repair of DSBs in human cells. Human lymphoblastoid cell lines TSCE5 and TSCE105 have, respectively, single and double I-SceI endonuclease sites in the endogenous thymidine kinase gene (TK) located on chromosome 17q. I-SceI expression generated DSBs at the TK gene. We used the novel transfection system (Amaxa Nucleofector) to introduce an I-SceI expression vector into the cells and randomly isolated clones. We found mutations involved in the DSBs in the TK gene in 3% of TSCE5 cells and 30% of TSCE105 cell clones. Most of the mutations in TSCE5 were small (1-30bp) deletions with a 0-4bp microhomology at the junction. The others consisted of large (>60) bp deletions, an insertion, and a rearrangement. Mutants resulting from interallelic HR also occurred, but infrequently. Most of the mutations in TSCE105, on the other hand, were deletions that encompassed the two I-SceI sites generated by NHEJ at DSBs. The sequence joint was similar to that found in TSCE5 mutants. Interestingly, some mutants formed a new I-SceI site by perfectly joining the two original I-SceI sites without deletion of the broken-ends. These results support the idea that NHEJ for repairing I-SceI-induced DSBs mainly results in small or no deletions. Thus, NHEJ must help maintain genomic integrity in mammalian cells by repairing DSBs as well as by preventing many deleterious alterations.
Honma
Masamitsu
M
Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. honma@nihs.go.jp <honma@nihs.go.jp>
Sakuraba
Mayumi
M
Koizumi
Tomoko
T
Takashima
Yoshio
Y
Sakamoto
Hiroko
H
Hayashi
Makoto
M
D001483:Base Sequence; D002460:Cell Line; D019457:Chromosome Breakage; D002874:Chromosome Mapping; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D017353:Gene Deletion; D005822:Genetic Vectors; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins
D029701:Saccharomyces cerevisiae Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific