| Abstract: | We examined the rejoining of noncomplementary restriction enzyme-produced DNA double-strand breaks in Escherichia coli and in cultured human cells. The enzymes used in this study, ClaI, BamHI and SalI, produce double-strand breaks with 5 protruding single strands. The joining of a ClaI-produced DNA end to a BamHI-produced end or to a SalI-produced end was examined at the DNA sequence level. End rejoining in E.coli was studied by transforming cultures with linear plasmid DNA that was gel purified from restriction digests, and end rejoining in cultured human cells was studied by introducing enzymes into the cells by electroporation. The human cells used contain an Epstein-Barr virus (EBV)-based shuttle vector, pHAZE, that was recovered and introduced into E.coli for further analysis. The major products of DNA end-joining processes observed in linear plasmid-transformed E.coli and in the human cells exposed to restriction enzymes were identical. Furthermore, the deletions observed in both systems and in the spontaneous mutant plasmids in untreated human cells had a common underlying feature: short stretches of directly repeated DNA at the junction sites. |
