Double-stranded break can be repaired by single-stranded oligonucleotides via the ATM/ATR pathway in mammalian cells.
Single-stranded oligonucleotide (SSO)-mediated gene modification is a newly developed tool for site-specific gene repair in mammalian cells; however, the corrected cells always show G2/M arrest and cannot divide to form colonies. This phenomenon and the unclear mechanism seriously challenge the future application of this technique. In this study, we developed an efficient SSO-mediated DNA repair system based on double-stranded break (DSB) induction. We generated a mutant EGFP gene with insertions of 24 bp to 1.6 kb in length as a reporter integrated in mammalian cell lines. SSOs were successfully used to delete the insertion fragments upon DSB induction at a site near the insertion. We demonstrated that this process is dependent on the ATM/ATR pathway. Importantly, repaired cell clones were viable. Effects of deletion length, SSO length, strand bias, and SSO modification on gene repair frequency were also investigated.
Wang
Zai
Z
Department of Biochemistry, The University of Hong Kong, Hong Kong SAR, People's Republic of China.
Zhou
Zhong-Jun
ZJ
Liu
De-Pei
DP
Huang
Jian-Dong
JD
D000483:Alleles; D064007:Ataxia Telangiectasia Mutated Proteins; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D004249:DNA Damage; D017931:DNA Primers; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D009841:Oligonucleotides / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases