T7 Dna Polymerase Rescues The Dna Loading Defect Of The A257t Linker Region Mutant Of T7 Helicase-Primase Protein

Bacteriophage T7 gp4 is a bifunctional primase‐helicase that oligomerizes into ring‐shaped hexamers around DNA in the presence of dTTP. The helicase and primase activities in T7 gp4 reside in two separate domains that are connected by a linker region. The linker region plays an important role in oligomerization and point mutations in the linker region have deleterious effects on the helicase function. In this study, we have investigated the biochemical and structural properties of linker region mutant A257T, which is analogous to the A359T mutant of the human mitochondrial DNA helicase protein Twinkle that is linked to human diseases. We show that A257T gp4 is highly defective in unwinding duplex DNA when measured by ensemble assays. Interestingly, we found that in single molecule unwinding assays, A257T gp4 was almost as active as WT gp4. We show that although A257T gp4 is normal in forming rings in the presence of dTTP, it is critically defective in loading onto the DNA. We discovered that T7 DNA polymerase rescued the DNA loading defect of A257T gp4. We propose that the inability to load on the DNA at efficient rates explains why A257T gp4 does not support T7 phage replication.