The combined DNA and RNA synthetic capabilities of archaeal DNA primase facilitate primer hand-off to the replicative DNA polymerase

Abstract

Replicative DNA polymerases cannot initiate DNA synthesis de novo and rely on dedicated RNA polymerases, primases, to generate a short primer. This primer is then extended by the DNA polymerase. In diverse archaeal species, the primase has long been known to have the ability to synthesize both RNA and DNA. However, the relevance of these dual nucleic acid synthetic modes for productive primer synthesis has remained enigmatic. In the current work, we reveal that the ability of primase to polymerize DNA serves dual roles in promoting the hand-off of the primer to the replicative DNA polymerase holoenzyme. First, it creates a 5'-RNA-DNA-3' hybrid primer which serves as an optimal substrate for elongation by the replicative DNA polymerase. Second, it promotes primer release by primase. Furthermore, modeling and experimental data indicate that primase incorporates a deoxyribonucleotide stochastically during elongation and that this switches the primase into a dedicated DNA synthetic mode polymerase.

Fig. 1. In vitro replication by PriSLX and PolB1-HE.

a Left panel: replication reactions containing 200 µM NTPs; 30 µM dNTPs; 2 µCi/10 µL α-³²P-dATP; 25 nM M13 ssDNA template; and 25 nM of protein(s) as indicated. 30 min at 75 °C. Right panel: representation of M13 ssDNA template. The suffix “PD”, Polymerase Dead, indicates versions of the primase and polymerase that have amino acid substitutions in the active site aspartate residues, D101,103A and D655,657A, respectively, that render them inactive. Two independent replicates of this experiment were performed. b Replication reactions with or without 200 µM NTPs as indicated; 30 µM dNTPs; 2 µCi/10 µL α-³²P-dATP; 25 nM M13 ssDNA template; and 25 nM of protein(s) as indicated. 30 min at 75 °C. Three independent replicates of this experiment were performed.

Fig. 2. DNA-synthetic mode PriSLX stimulates RNA-primer extension.

a–c Primer elongation reactions with 100 µM dNTPs; 25 nM 5′-cy5-labeled primer as indicated annealed to 80 nt template; PolB1-HE titration (25, 12.5, 6.25 nM) as indicated; 25 nM PriSLX as indicated; and PriSLX PD (Polymerase Dead) as indicated. Reactions were incubated for 5 min at 75 °C prior to termination and electrophoresis on denaturing polyacrylamide gels. Three independent replicates of this experiment were performed. d K_d determination by fluorescence polarization. Titration series of PolB1-HE as indicated with indicated 5′-Cy5-labeled primer annealed to 80 nt template. Data points indicate the mean value and error bars represent standard deviation. Four independent replicates of this experiment were performed.

Fig. 3. Primer elongation by sub-saturating amounts of PolB1-HE.

Primer elongation reactions of 100 µM dNTPs; 25 nM 5′-cy5-labeled primer as indicated annealed to 80 nt template; 6 nM PolB1-HE as indicated; BSA titration (200, 100, 50, 25 nM) and control (200 nM) as indicated; PriSLX titration (200, 100, 50, 25 nM) and control (200 nM) as indicated; and PriSLX PD (Polymerase Dead) titration (200, 100, 50, 25 nM) and control (200 nM) as indicated. Reactions were incubated for 5 min at 75 °C prior to termination and electrophoresis on denaturing polyacrylamide gels. Three independent replicates of this experiment were performed.

Fig. 4. PriSLX elongation activity.

a Primer elongation reactions of 100 µM dNTPs; 25 nM 5′-cy5-labeled primer as indicated annealed to 80 nt template; 25 nM PriSLX as indicated; BSA titration (25, 12.5 nM) and control (25 nM) as indicated; and PolB1-HE PD (Polymerase Dead) titration (25, 12.5 nM) and control (25 nM) as indicated. Reactions were incubated for 5 min at 75 °C prior to termination and electrophoresis on denaturing polyacrylamide gels. Three independent replicates of this experiment were performed. b Primer elongation reactions of 100 µM NTPs or dNTPs as indicated; 25 nM 5′-cy5-labeled primer annealed to 80 nt template; and 25 nM PriSLX. Reaction times as indicated at 75 °C prior to termination and electrophoresis on denaturing polyacrylamide gels. Three independent replicates of this experiment were performed. c Primer elongation reactions of 100 µM NTPs or dNTPs as indicated; 25 nM 5′-cy5-labeled primer as indicated annealed to 80 nt template; and PriSLX titration (50, 25, 12.5 nM) as indicated. Reactions were incubated for 5 min at 75 °C prior to termination and electrophoresis on denaturing polyacrylamide gels. Four independent replicates of this experiment were performed.

Fig. 5. Binding and kinetic analyses of PriSLX.

a K_d determination by fluorescence polarization. Titration series of PriSLX as indicated with indicated 5′-cy5-labeled primer annealed to 80 nt template. Data points indicate the mean and error bars represent standard deviation. Four independent replicates of this experiment were performed. b Steady-state kinetic analysis of PriSLX to determine initial rate of product formation (K_obs) and steady-state turnover rate (K_ss). Single nucleotide elongation reactions with GTP or dGTP as indicated and 5′-cy5-labeled 19 nt RNA-primer annealed to 80 nt template. Reactions timed by rapid quench flow as indicated; 60 °C. Data points indicate the mean and error bars represent standard deviation. Two independent replicates of this experiment were performed.

Fig. 6. Impact of dNMP incorporation on primer synthesis.

a Geometric probability model of dNMP incorporation by PriSLX as a function of primer length. Model assumes NTP as the initiating nucleotide and that nucleotide incorporation proceeding from RNA-primer is governed exclusively by equal weighting of relative dNTP/NTP abundance and elongation-site binding affinity. b Top panel: representation of the 5′-triphosphate 8 nt RNA-primer annealed to the poly-(24 nt T)-track. Bottom panel: primer elongation reactions of ATP (2 µCi/20 ul α-³²P-ATP and 165 nM ATP) or dATP (2 µCi/20 ul α-³²P-dATP and 165 nM dATP) as indicated; 15 nM 5′-triphosphate 8 nt RNA-primer annealed to poly-(24 nt T)-track template; and 3.75 nM PriSLX as indicated. Reactions were incubated for 2 min at 75 °C prior to termination and electrophoresis on denaturing polyacrylamide gels. Four independent replicates of this experiment were performed. c Primer elongation reactions of 2 µCi/20 ul α-³²P-ATP with unlabeled ATP and dATP as indicated for total nucleotide concentration as indicated; 15 nM 5′-triphosphate 8 nt RNA-primer annealed to poly-(24 nt T)-track template; and 3.75 nM PriSLX as indicated. Reactions were incubated for 2 min at 75 °C prior to termination and electrophoresis on denaturing polyacrylamide gels. Three independent replicates of this experiment were performed.

Fig. 7. Model of the primer hand-off.

After primase initiates primer synthesis by RNA-synthesis, a favored stochastic deoxyribonucleotide incorporation switches primase to DNA-synthesis. This promotes primase disengagement from the primer and subsequent hand-off to replicative polymerase. a, b A scenario of earlier or later switch to DNA-synthesis, respectively, by primase. c While unfavored, a scenario where primase does not switch to DNA-synthesis. As a back-up failsafe, the caliper mechanism constrains RNA-primer length, halting synthesis. With synthesis halted, primase dissociates from the primer.