【作者】 吴雪松； 辛立； 殷文璇； 尚曦莹； 陆璐； RoryM.Watt； KathrynS.E.Cheah； 黄建东； 刘德培； 梁植权；

【Author】 Xue-Song Wu, Li Xin, Wen-Xuan Yin, Xi-Ying Shane, Lu Lu, Rory M. Watt, KathrynS.E.Cheah, Jian-Dong Huang, De-Pei Liu, Chih-Chuan Liang(National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College (PUMC), Beijing 100005, P. R. China and Department of Biochemistry, The University of Hong Kong, /F Laboratory Block, Faculty of Medicine Building, 21 Sasson Road,Pokfulam, Hong Kong SAR, China

【机构】 中国医学科学院中国协和医科大学医学分子生物学国家重点实验室； 香港大学生化系； National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College (PUMC),Beijing 100005,P. R. China；

【摘要】 <正>Single-stranded oligonucleotide (SSO)-mediated gene modification provides a potential strategy for studies in genomic engineering and attempts in gene therapy. It has been shown competent to modify target genes in microorganisms and mammalian cells, with varying reproducibility and unknown mechanisms. Of all the impediments in SSOs’ way, chromatin environments in host cells possess important but not exactly deciphered impact The fact that chromatin structure changes in periodic cell cycles suggests us to systematically investigate SSO-mediated gene repair at various stages of cell cycle progression in mammalian cells. We found that S phase when DNA replicated provided an opportunistic window for SSO’s activity. We observed that the activity of SSO was particularly elevated in the thymidine-treated cells to a frequency approximate 10-fold of that in untreated controls. And the increase correlated positively with the duration of SSO/thymidine co-incubation with host cells. Further studies demonstrated mat the increased level of SSO-mediated gene repair arose from a thymidine-induced delay of replication fork progression, which meant the replication fork progressed very slowly, while not totally stopped. Our findings point to a mechanism for SSO-mediated gene repair whereby the slowed progression of replication forks might enable SSOs to stably anneal with their target regions of single-stranded DNA and incorporate into genome DNA as nascent strand. These studies provide a new approach for manipulating SSO-mediated repair and directing how its efficiency may be significantly enhanced within mammalian cells.更多还原

【Abstract】 Single-stranded oligonucleotide (SSO)-mediated gene modification provides a potential strategy for studies in genomic engineering and attempts in gene therapy. It has been shown competent to modify target genes in microorganisms and mammalian cells, with varying reproducibility and unknown mechanisms. Of all the impediments in SSOs’ way, chromatin environments in host cells possess important but not exactly deciphered impact The fact that chromatin structure changes in periodic cell cycles suggests us to systematically investigate SSO-mediated gene repair at various stages of cell cycle progression in mammalian cells. We found that S phase when DNA replicated provided an opportunistic window for SSO’s activity. We observed that the activity of SSO was particularly elevated in the thymidine-treated cells to a frequency approximate 10-fold of that in untreated controls. And the increase correlated positively with the duration of SSO/thymidine co-incubation with host cells. Further studies demonstrated mat the increased level of SSO-mediated gene repair arose from a thymidine-induced delay of replication fork progression, which meant the replication fork progressed very slowly, while not totally stopped. Our findings point to a mechanism for SSO-mediated gene repair whereby the slowed progression of replication forks might enable SSOs to stably anneal with their target regions of single-stranded DNA and incorporate into genome DNA as nascent strand. These studies provide a new approach for manipulating SSO-mediated repair and directing how its efficiency may be significantly enhanced within mammalian cells.更多还原