1. Figure S1 RNA-primed DNA synthesis by T7 DNA polymerase. DNA synthesis on an M13 ssDNA template catalyzed by T7 DNA polymerase requires primers annealed to the template. The primer was either synthesized de novo from ATP and CTP by gene 4 protein (A) or supplied as 5'-ACCC-3' (B). Reaction mixtures contained dGTP, dTTP, dATP, [  -  P]dCTP, M13 ssDNA, T7 DNA polymerase, and 0, 7, 22, 66, 200 nM of the indicated gene 4 protein. After incubation for 5 min at 37 °C, the products of the reaction were spotted on DE81 membrane, and unincorporated dNTP was removed by extensively washing with ammonium formate. The amount of DNA synthesis was determined by measuring the amount of radioactive dCMP adsorbed to the DE81 membrane.

2. Figure S2 Helicase activity of the altered gene 4 proteins. (A) DNA unwinding activities of the gene 4 protein were measured by monitoring the amount of ssDNA displaced from the duplex DNA substrate. The DNA substrate used for measuring helicase unwinding activity consists of a 32 P labeled 45-mer oligonucleotide annealed to a 65-mer oligonucleotide; 20 nucleotides at the 3'-end of the 45-mer oligonucleotide are not complementary to the 65-mer oligonucleotide. The DNA substrate (100 nM) was incubated with 0.5 mM dTTP and the indicated amounts of each gene 4 protein. After 5 min of incubation at 37 °C, the amount of 5'- 32 P radiolabeled 45-mer oligonucleotide displaced from the 65-mer oligonucleotide was determined by non-denaturing PAGE and phosphorimage analysis. (B) DNA binding assay, DNA unwinding assay, and dTTP hydrolysis assay with the indicated gene 4 proteins were performed as described.

3. wt-gp4 gp4-link(del6) Figure S3 Oligoribonucleotide synthesis in the presence of different substrate concentrations. (A) Template-directed de novo synthesis. Increasing amounts of ATP were added to the indicated gp4 and [  - 33 P]CTP in the presence of template containing the recognition sequence 5'-GGGTC-3' for 30 min at 37 °C. The reaction products were analyzed on 25% denaturing polyacrylamide gel. (B) Template-directed AC extension. Increasing amounts of AC were added to the indicated gp4 and [  - 33 P]CTP in the presence of template containing the recognition sequence 5'-GGGTC-3' for 30 min at 37 °C. The reaction products were analyzed on 25% denaturing polyacrylamide gel. AB

4. wt-gp4 gp4-link(del6) pppACCC pppAC Figure S4 Template-directed de novo oligoribonucleotide synthesis. Increasing amounts of ATP were added to the indicated gp4 and [  - 33 P]CTP in the presence of template containing the recognition sequence 5'-GGGTC-3' (A) or only the core recognition sequence 5’-GTC-3’ (B) for 20 min at 37 °C. The reaction products were analyzed on 25% denaturing polyacrylamide gel. Reaction products are indicated to the left of the gel. AB

5. Figure S5 Oligoribonucleotide synthesis catalyzed by different gp4 protein mixtures. A fixed amount (200 nM) of the indicated gene 4 protein was mixed with the 56 kDa- gp4/D237A or with buffer. The mixture was incubated at room temperature for 10 min and then with DNA template 5'-GGGTCAAAAAAAAAA-3' in the standard de novo synthesis reaction with ATP and [  - 33 P]CTP (top) or in the standard AC extension assay with AC and [  -33P]CTP (bottom) for 30 min at 37°C. Reaction products were analyzed on a 25% denaturing polyacrylamide gel. The major products, 5'-pppACCC- 3‘, and 5'-ACCC-3’, are indicated to the left of the gel. 1 2 3 4 5 6 7 8 9 10 pppACCC ACCC 1- 56 kDa-gp4 2- 56 kDa-gp4/D237A 3- wt-gp4 4- wt-gp4 + 56 kDa-gp4/D237A 5- wt-gp4/D237A 6- wt-gp4/D237A + 56 kDa-gp4/D237A 7- gp4-link(del6) 8- gp4-link(del6) + 56 kDa-gp4/D237A 9- gp4-link(del6)/D237A 10- gp4-link(del6)/D237A + 56 kDa-gp4/D237A