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. 2016 Oct 24;291(51):26566–26585. doi: 10.1074/jbc.M116.753160

FIGURE 9.

FIGURE 9.

RNase H cleavage during processive nucleotide incorporation. A, RNase H activities during processive nucleotide incorporation were explored by performing RNA cleavage assays in the presence of all 4 nucleotides (100 μm). The procedures were the same as described previously except that data were collected for 20 s to allow full extension and multiple rounds of cleavage to occur. To avoid overlapping bands for extension and cleavage products, the two activities were assayed in parallel as described above. Positions for the primer (d25), primer extended by one nucleotide (d26), and fully extended product (d45) were marked. The same nomenclature as used in Fig. 8 was applied here to label positions of full-length RNA template (0) and cleaved products. Positions for the longest cleavage products formed during each round of degradation were marked as +7, +16, +20, and +25. The numbers of nucleotides incorporated before each round of RNA degradation were also labeled as 9, 4, and 5, respectively. B, time courses of processive nucleotide incorporation were simulated according to Scheme 3, with smooth lines indicating the best fit. Data within the first 1 s were shown to better display the main content of the data and avoid the long non-zero tails of fitting curves. The fitted rates for sequential polymerization steps were 94 ± 6, 19 ± 1, 33 ± 3, 30 ± 4, 19 ± 2, 35 ± 8, 36 ± 10, 43 ± 15, 27 ± 6, 29 ± 8, 20 ± 5, 23 ± 7, 36 ± 18, 21 ± 7, 26 ± 12, 21 ± 10, 29 ± 20, 36 ± 34, 20 ± 13, and 10 ± 5 s−1, respectively. The apparent incorporation rates for +N8, +N4, and +N5 were then determined as 3.4 ± 0.3, 6.3 ± 1.3, and 5 ± 1.7 s−1, respectively. C, the apparent incorporation rates obtained from panel B were used in the global analysis of time courses of RNA cleavage according to Scheme 4. Apparent incorporation rates and fitted rates for each round of RNA degradation are summarized in Scheme 4 and Table 3. Color codes: red, 45-nt RNA template; green, 1st cleavage (38 nt); blue, 2nd cleavage (29 nt); yellow, 3rd cleavage (25 nt); cyan, fourth cleavage (20-nt).