Figure 3.

Phosphate removal from a 3′-P gap. (A) Schematic representation of the enzymatic reactions involved in the repair of a 3′-P harbouring gap. Initial substrate is a gap surrounded by 5′-P and 3′-P DNA ends (i). The 3′ phosphate terminus is converted into a 3′-OH terminus by a 3′ phosphatase (I) resulting in a gap (ii) that can be filled by a DNA polymerase (II, iii) and ligated (III) to yield a 34mer (iv). (B) The ³²P-labelled substrate (i) was incubated at 37°C for 15 min with 0.5 (lanes 2, 6 and 10), 1 (lanes 3, 7 and 11), 1.5 (lanes 4, 8 and 12) or 3 μg (lanes 5, 9 and 13) of WCE proteins from the indicated cell lines. Products were separated by denaturing PAGE. (C) 3′-P harbouring gap substrate (i) was incubated with 1 μg of WCE of the indicated cell lines at 37°C and aliquots of each reaction were removed at the times indicated. The resulting products were separated by denaturing PAGE and the conversion of the 3′-P into 3′-OH was quantified.