Strand displacement DNA synthesis by DNA polymerase gp90 exo- of Pseudomonas aeruginosa phage 1

Abstract

Strand displacement DNA synthesis is essential for DNA replication. Gp90, the sole DNA polymerase of Pseudomonas aeruginosa phage 1, can bypass multiply DNA lesions. However, whether it can perform strand displacement synthesis is still unknown. In this work, we found that gp90 exo^- could perform strand displacement synthesis, albeit its activity and processivity were lower than those of primer extension. Gp90 exo^- itself could not unwind Y-shaped or fork DNA. Tail and gap at DNA fork were necessary for efficient synthesis. High GC content obviously inhibited strand displacement synthesis. Consecutive GC sequence at the entrance of fork showed more inhibition effect on DNA synthesis than that in the downstream DNA fork. The fraction of productive polymerase and DNA complex (A values) was higher for fork than gap; while their average extension rates (k_p values) were similar. However, both A and k_p values were lower than those for the primer/template (P/T) substrate. The binding of gp90 exo^- to fork was tighter than P/T or gap in the absence of dATP. In the presence of dATP to form ternary complex, the binding affinity of gp90 exo^- to P/T or gap was increased compared with that in the binary complex. Abasic site, 8-oxoG, and O⁶-MeG inhibited and even blocked strand displacement synthesis. This work shows that gp90 exo^- could perform strand displacement DNA synthesis at DNA fork, discovering the presence of new functions of PaP1 DNA polymerase in DNA replication and propagation of PaP1.

Keywords: Binding affinity determined by surface plasmon resonance; DNA fork; DNA lesion or damage; Fast kinetic analysis; GC percentage or sequences.