Figure 6.

Binding of RIP60 GST-Z2 to the USR and DSR enhances ligation of linear pCH127. A ligation enhancement assay was used to test for the ability of RIP60 fusion proteins to mediate DNA looping between the USR and DSR. (A) pCH127 contains a 1.1 kb fragment of the DHFR ori-β region that includes both the USR and DSR RIP60 binding sites. (B) pCH127 was linearized with AccI and incubated with the indicated protein factors for 15 min prior to the addition of ligase and ATP for 5 min. The products of the reactions were resolved by gel electrophoresis, blotted to nylon, and visualized by hybridization with ³²P-labeled pCH127. Ligation products representing open circles or linear dimers are indicated by the square bracket. Addition of DSR competitor DNA eliminated increased ligation by GST-Z2 (lane 9) whereas the AT-rich region from the SV40 origin of replication had no effect (lane 10). Dephosphorylated linear pCH127 did not support ligation with (lane 11) or without (not shown) GST-Z2.