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. 2019 Sep 23;47(20):10693–10705. doi: 10.1093/nar/gkz792

Figure 5.

Figure 5.

The polar residue reduces rCTP incorporation by MsDpo4. The results of primer extension assays conducted using different concentrations of wt- MsDpo4 and variants (MsDpo4-L14Y, MsDpo4-C47T & MsDpo4-C47T+L14Y) at different concentrations of rCTP or dCTP are shown. The concentrations of enzyme used were 5 nM (Lanes 1, 2 and 3), 10 nM (Lanes 4, 5 and 6) and 15 nM (lanes 7, 8 and 9). The concentrations of incoming nucleotide (rCTP or dCTP) used were 1 μM (lanes 1, 4 and 7), 2 μM (lanes 2, 5, and 8) and 4 μM (lanes 3, 6 and 9). The assays show that mutation of Cys47 to Thr enhances the ability of MsDpo4 to exclude ribonucleotides. The MsDpo4-C47T and MsDpo4-C47T+L14Y proteins show higher selectivity for dCTP than MsDpo4-WT or MsDpo4-L14Y. These experiments shows that engineered residue (Thr47) enhances sugar selectivity.