Fig. 9. XKu86 and RP-A are required for ligation of dsDNA fragments on DNA beads incubated with HSS. Analysis of duplex DNA ligation by agarose gel electrophoresis and autoradiography. DNA bound to magnetic beads was incubated with ³²P-end labeled pUC19 in the presence of mock depleted, RP-A-depleted, XKu86-depleted HSS extracts or with a buffer for 1 hour at 23°C. After incubation, the DNA beads were extensively washed with increasing concentrations of salt (up to 2 M NaCl) and detergent (up to 1% SDS). DNA beads were then equilibrated with BglII restriction enzyme buffer and digested with BglII (5 units per 100 ng DNA) for 2 hours at 37°C. The DNA fragments released from the beads were run on a 0.8% agarose gel. (A) Autoradiography: the labeled DNA fragments released by BglII digest and the linearized pUC19 are indicated on the side. (B) Ethidium bromide staining of the gel. Lane 1, mock depleted extracts; lane 2, RP-A-depleted extracts; lane 3, XKu86-depleted extracts; lane 4, buffer. Molecular weight standards are indicated on the left-hand side. The depletion of XKu86 and, to a lesser extent, RP-A from the extract inhibits end to end ligation of linear DNA fragments (arrows indicate the 7.7 kbp DNA fragments, arrowheads indicate the 5.7 kbp fragment). The asterisks indicate the presence of a contaminating DNA band migrating at 6.8 kbp. Ethidium bromide staining of the gel served as a control measure of the loading. In lane 2 in (B), the presence of the band migrating more rapidly than the 3 kbp fragment might be due to the star activity of BglII.

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