FIG. 4.

Coding joints from wild-type and Ku70^−/− cells. A. PCR strategy for cloning sequences resulting from RAG-induced NHEJ, HDR, or SSA in the DRGFP-CE reporter. Because of the large decrease in NHEJ in Ku70^−/− cells, the amplified repair product is much lower than that from wild-type cells but is restored by Ku70 expression. Genomic DNA is predigested with NdeI and MfeI, which cleave within the unrearranged reporter. Primers 1 and 2 are used to amplify the RAG-induced repair products. The PCR product is inserted into a TA vector and transformed into E. coli. Plasmids containing an insert are subjected to BcgI digestion, which will cleave the insert if HDR or SSA occurs, but not if NHEJ occurs. DNA sequencing was performed on inserts derived from NHEJ. Symbols are as in Fig. 1. B. Ku70 deficiency alters the spectrum of coding joint products. Although some of the coding joints from Ku70^−/− cells resemble those found in wild-type cells, most of the coding joints have larger deletions or other modifications. Microhomology (underlined), N nucleotides (red), P nucleotides (green), and an internally deleted nucleotide (black box) are indicated. Abbreviations: 1, DRGFP1; 2, DRGFP2.