Investigating The Role of the Primer‐Template Terminus in the Conformational Dynamics of Bacillus stearothermophilus DNA Polymerase I Using FRET
Authors: Christian Thomas David , Konigsberg William 
Published: November 2023

We have monitored DNA polymerase‐DNA interactions via FRET at the ensemble and single‐molecule level in the presence of dideoxy (ddp/t) and deoxy (dp/t) terminated primer‐templates. FRET was monitored by placing Cy3B eight base pairs from the p/t terminus and Atto647N on the tip of the fingers subdomain using a D695C mutant of B. stearothermophilus DNA polymerase I large fragment (Bst pol). Bulk‐solution titrations showed the FRET efficiency in the binary complex to be ~0.70; consistent with crystallographic data. Pre‐steady state kinetic analysis showed that the deoxynucleotide triphosphate (dNTP) selectivity of the labeled Bst pol was not affected. In bulk‐solution, there were different FRET changes upon the addition of a dNTP when using a ddp/t vs a dp/t. Titrations with increasing [dNTP] using a ddp/t indicated that the two dyes moved closer together, consistent with the previously observed open‐to‐closed conformational change that occurs upon dNTP binding. For the ddp/t, the apparent dissociation constants (Kd(app)) indicated that this movement is relatively insensitive to the identity of the incoming dNTP; the correct dNTP having a Kd(app) = 8.43 ± 0.80 μM versus a Kd(app) = 69.7 ± 6.7 μM for an incorrect incoming dNTP. In contrast, when a dp/t was used, the fingers domain adopted a more open conformation upon the addition of correct or incorrect dNTPs. For the dp/t, the Kd(app) for correct vs. incorrect dNTP binding were markedly different; with the correct dNTP having a Kd(app) = 30.2 ± 4.9 μM and the incorrect dNTP having a Kd(app) > 1 mM.

Single‐molecule measurements showed that the binary FRET efficiency, observed at the ensemble level, was composed of two states; a high‐FRET (~0.80) and a low‐FRET (~0.25) state. When a ddp/t was used, the low FRET state in the binary complexes predominates; with greater than 95% occupancy. With the ddp/t, a transition from the low‐FRET state to two higher‐FRET states (~0.35 and ~0.60) was observed with the correct dNTP; corresponding to the previously observed ajar and closed states. There was a small change in FRET in the presence of 1 mM incorrect dNTP. When the dp/t was used, 60% of the binary population adopted the low FRET state and 40% adopted the high FRET state. With the dp/t, a transition from the low FRET state to two higher FRET states was also observed in the presence of the correct dNTP. With the incorrect dNTP approximately 50% of the molecules adopted the ajar state and 50% adopted the high FRET state. When a dp/t containing 4 mismatched base‐pairs was used, the binary complexes adopted two FRET states; one between the ajar and closed states (~0.50) and another corresponding to the previously observed high‐FRET state (~0.80).

These results suggest that the 3′ hydroxyl group, and possibly the metal ion with which it coordinates in the ternary structure, may play a key role in the conformational changes in the fingers subdomain associated with dNTP selection and incorporation. The absence of the high‐FRET state with the ddp/t indicates that the 3′ hydroxyl is necessary for transfer of the primer from the polymerase to the exonuclease site. The two states observed with the mismatched dp/t may correspond to the fidelity‐checking and exonuclease states previously observed for pol I.

Support or Funding Information

Supported by NIH Grant GM 063276‐09