DNA Polymerase-Parental DNA Interaction Is Essential for Helicase-Polymerase Coupling during Bacteriophage T7 DNA Replication

Abstract

DNA helicase and polymerase work cooperatively at the replication fork to perform leading-strand DNA synthesis. It was believed that the helicase migrates to the forefront of the replication fork where it unwinds the duplex to provide templates for DNA polymerases. However, the molecular basis of the helicase-polymerase coupling is not fully understood. The recently elucidated T7 replisome structure suggests that the helicase and polymerase sandwich parental DNA and each enzyme pulls a daughter strand in opposite directions. Interestingly, the T7 polymerase, but not the helicase, carries the parental DNA with a positively charged cleft and stacks at the fork opening using a β-hairpin loop. Here, we created and characterized T7 polymerases each with a perturbed β-hairpin loop and positively charged cleft. Mutations on both structural elements significantly reduced the strand-displacement synthesis by T7 polymerase but had only a minor effect on DNA synthesis performed against a linear DNA substrate. Moreover, the aforementioned mutations eliminated synergistic helicase-polymerase binding and unwinding at the DNA fork and processive fork progressions. Thus, our data suggested that T7 polymerase plays a dominant role in helicase-polymerase coupling and replisome progression.

Efficacy in Galleria mellonella Larvae and Application Potential Assessment of a New Bacteriophage BUCT700 Extensively Lyse Stenotrophomonas maltophilia

“…The genomic annotation of phage BUCT700 showed that 7 ORFs were associated with replication and transcription, including DNA primase/helicase (ORF 20), DNA helicase (ORF 21), DNA polymerase I (ORF 22), 5′–3′ exonuclease (ORF 24), DNA exonuclease (ORF 25), exonuclease (ORF 26), and DNA-dependent RNA polymerase (ORF 34). During DNA replication, a DNA helicase and a DNA polymerase cooperatively unwind the parental DNA ( 29 ). Although DNA polymerase I (ORF 22) has exonuclease activity, phage BUCT700 still carries 3 genes (ORF 24, 25, and 26) encoding exonucleases.…”