Figure 3. Rad54-AA is defective in strand invasion and primer extension activities.

A. Schematic of the MAT chromosomal locus used for the examination of DNA repair synthesis. Arrows depict the direction of primers used for detection of primer extension intermediates by PCR. B. The primer extension step of recombination is compromised in the rad54 PIP-box mutant. The top panel shows the formation pA-pB product, which results from minimal DNA synthesis from the invading strand. Samples were taken at 1, 2 or 5 h after HO endonuclease cutting. The bottom panel shows pC-pF control product. C. Rad54-AA is defective in DNA repair synthesis in vitro. Rad51 and DNA substrates were pre-formed into nucleoprotein filaments as described, then either Rad54 wild type (wt, lanes 1–7) or Rad54-AA (lanes 8–14) was incorporated and D-loop formation was initiated. DNA synthesis reactions were then performed using Polymerase δ (15 nM), and increasing concentrations (2.5, 5, 10, 20 nM) of the PCNA clamp, with or without the PCNA clamp loader, RFC (10 nM), in the presence of RPA (666 nM). The reactions were monitored using labeled α-[³²P]-dATP, and percentage of each reaction product shown below.