New mammalian cellular systems to study mutations introduced at the break site by non-homologous end-joining.
The non-homologous end-joining (NHEJ) pathway is a mechanism to repair DNA double strand breaks, which can introduce mutations at repair sites. We constructed new cellular systems to specifically analyze sequence modifications occurring at the repair site. In particular, we looked for the presence of telomeric repeats at the repair junctions, since our previous work indicated that telomeric sequences could be inserted at break sites in germ-line cells during primate evolution. To induce specific DNA breaks, we used the I-SceI system of Saccharomyces cerevisiae or digestion with restriction enzymes. We isolated human and hamster cell lines containing the I-SceI target site integrated in a single chromosomal locus and we exposed the cells to a continuous expression of the I-SceI endonuclease gene. Additionally, we isolated human cell lines that expressed constitutively the I-SceI endonuclease and we introduced the target site on an episomal plasmid stably transfected into the cells. These strategies allowed us to recover repair junctions in which the I-SceI target site was modified at high frequency (100% in hamster cells and about 70% in human cells). Finally, we analyzed junctions produced on an episomal plasmid linearized by restriction enzymes. In all the systems studied, sequence analysis of individual repair junctions showed that deletions were the most frequent modifications, being present in more than 80% of the junctions. On the episomal plasmids, the average deletion length was greater than at intrachromosomal sites. Insertions of nucleotides or deletions associated with insertions were rare events. Junction organization suggested different mechanisms of formation. To check for the insertion of telomeric sequences, we screened plasmid libraries representing about 3.5 x 10(5) junctions with a telomeric repeat probe. No positive clones were detected, suggesting that the addition of telomeric sequences during double strand break repair in somatic cells in culture is either a very rare event or does not occur at all.
Rebuzzini
Paola
P
Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, 27100 Pavia, Italy.
Khoriauli
Lela
L
Azzalin
Claus M
CM
Magnani
Elisa
E
Mondello
Chiara
C
Giulotto
Elena
E
D000818:Animals; D001483:Base Sequence; D006224:Cricetinae; D004249:DNA Damage; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010957:Plasmids; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D016615:Telomere / Q000235:genetics*
D029701:Saccharomyces cerevisiae Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific