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. 2007 Feb 8;26(4):1010–1023. doi: 10.1038/sj.emboj.7601559

Figure 1.

Figure 1

Template-independent polymerase activity of pol mu on substrates in free solution. (A) Two 73 bp substrates with 3′ overhangs were used to test for pol mu template-independent polymerase activity in free solution. An asterisk indicates the position of the radioisotope label. (B, C) In each reaction, 20 nM substrate was incubated with the protein(s) indicated above each lane in a 10 μl reaction for 1 h at 37°C. After incubation, reactions were deproteinized and analyzed using 11% denaturing PAGE. Protein concentrations: Ku, 25 nM; X4-LIV, 50 nM; pol mu, 25 nM, where X4-LIV refers to XRCC4:DNA ligase IV. The specified dNTP was added to 100 μM. ‘dN' means that all the four dNTPs (100 μM each) were included. No ATP was added, unless specified. Template-independent polymerase synthesis results in extension of the radiolabeled strand, and hence, the more slowly moving species located above the substrate band.