Supplementary material for George et al. (2001) Proc. Natl. Acad. Sci. USA 98 (15), 8290-8297. (10.1073/pnas.131007598)
Fig. 9.
Analysis of repair products by using a unique PacI site in the breakable plasmid. In this experiment, the homologous plasmid was pJG10, and the breakable plasmid was pJG4P, a derivative of pJG4 with a unique PacI site cloned into the vector portion. In A, predicted repair products are depicted with increasing numbers of pJG10 copies sandwiched in between copies of pJG4P. In B, DNA was prepared from JG99S cells containing the two plasmids either without infection (0 min) or after 5, 10, 15, 20, or 30 min of infection by T4 K10. Total nucleic acids were prepared as described by George and Kreuzer (1), digested with PacI, and separated by inverting-field gel electrophoresis. The digest in each even-numbered lane also contained HaeIII, to test whether the products had been replicated throughout their length. The Southern blot was hybridized with a probe made from the vector portion of pJG4 (Left) or the "a" fragment of pJG10 (Right). Molecular size markers consisting of intact l DNA and restriction fragments of l were also run, allowing assignment of the repair products by size (data not shown). A series of repair products was evident with increasing numbers of homologous plasmid (pJG10) between copies of breakable plasmid (pJG4P) (positions indicated by n = 1, n = 2, etc., with n being the number of homologous plasmid copies embedded between breakable plasmid DNA). On the Left, this series had a distinct band at n = 4 and then a more intense band, just above, which would include n = 5 and all higher ns (this was the position where the resolution of the inverting-field gel ended). Note that the intensity of the bands with increasing n decreases more dramatically in the Left than in the Right, which is expected because the probe in the Left would hybridize only to the pJG4P vector DNA at the ends of the sandwiches, whereas the probe in the Right would hybridize equally to all homologous segments throughout the sandwiches. Additional repair product consisted of PacI-linearized pJG4P, either with or without coconversion of the b marker [indicated at the Bottom Right; note that a doublet of bands, because of the presence or absence of coconversion, is also evident for at least the first (n = 1) sandwich]. The PacI-linearized repaired DNA may arise from monomer circle products and/or from multimeric pJG4P product. Note that these products are completely resistant to HaeIII and therefore replicated throughout their length by the T4 machinery. These products migrate significantly slower than substrate (unreplicated) pJG4P DNA because of the glucosyl modifications (the aB product is also 227 bp larger). The very intense HaeIII-sensitive band near the Bottom Left is PacI-linearized pJG4P DNA (that had escaped I-TevI cutting in vivo). The HaeIII-sensitive band in the Right, indicated by the arrow, is unreplicated pJG10 DNA (circular form). We do not have a firm assignment for the band indicated with an asterisk on the Right. It hybridizes only to the "a" probe, not pJG4P vector, and so it is presumably a form of pJG10 DNA. It is significantly increased in the HaeIII digest but present at a lower level even without HaeIII. The most likely explanation is that this band consists of replicated nicked circular pJG10 DNA, and that a nicking activity in the HaeIII generated more of this product from replicated supercoiled circular pJG10 DNA.Reference:
1. George, J. W. & Kreuzer, K. N. (1996) Genetics 143, 1507-1520.