DNA Helicase–Polymerase Coupling in Bacteriophage DNA Replication

Abstract

Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase cooperatively unwind the parental DNA. By surveying recent data from three bacteriophage replication systems, we summarized the mechanistic basis of DNA replication by helicases and polymerases. Kinetic data have suggested that a polymerase or a helicase alone is a passive motor that is sensitive to the base-pairing energy of the DNA. When coupled together, the helicase–polymerase complex is able to unwind DNA actively. In bacteriophage T7, helicase and polymerase reside right at the replication fork where the parental DNA is separated into two daughter strands. The two motors pull the two daughter strands to opposite directions, while the polymerase provides a separation pin to split the fork. Although independently evolved and containing different replisome components, bacteriophage T4 replisome shares mechanistic features of Hel–Pol coupling that are similar to T7. Interestingly, in bacteriophages with a limited size of genome like Φ29, DNA polymerase itself can form a tunnel-like structure, which encircles the DNA template strand and facilitates strand displacement synthesis in the absence of a helicase. Studies on bacteriophage replication provide implications for the more complicated replication systems in bacteria, archaeal, and eukaryotic systems, as well as the RNA genome replication in RNA viruses.

The N-terminal domain of the human mitochondrial helicase Twinkle has DNA binding activity crucial for supporting processive DNA synthesis by Polymerase γ

“…Thus, concomitant DNA synthesis by Polγ can partially restore the unwinding defect caused by NTD deletion. Such cooperativity has been observed in T7 replisome, where the DNA polymerase accelerates the unwinding activity of T7 gp4 (34,35,39).…”

“…Because the ATPase site lies at the subunit interface, the formation of oligomer would be sufficient for ATP hydrolysis. On the other hand, the helicase function requires coordination between DNA binding-release steps and ATP binding-hydrolysis steps in each subunit (34,35). Hence, any defect in DNA binding activity can decouple the ATPase activity from the motor function resulting in poor helicase function.…”

“…1). Accordingly, we cloned the sequence corresponding to amino acid residues 43-372 to make the NTD construct (35,596 Da) lacking the linker domain; the 1-42 aa is the predicted mitochondrial targeting sequence. This NTD construct was reported in a previously published study of Twinkle (19), while the minimal CTD construct (36,650 Da) that contained amino acids from 360-684, which includes the CTD and the linker region only, is novel.…”

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

“…This interaction may help orient the parental DNA for synergistic separation by gp4 and gp5. A similar positively charged cleft can be mapped in B-family T4/RB69 and Φ29 polymerases [28]. Moreover, with additional domains (TPR2) encircling the single-stranded DNA on the leading strand, Φ29 polymerase can catalyze efficient strand-displacement synthesis without a helicase [42][43][44].…”

“…The helicase enhances polymerase processivity and activity, whereas the polymerase stimulates helicase unwinding [8]. Both the helicase and polymerase can translocate on ssDNA; however, the helicase alone can only unwind dsDNA inefficiently with frequent backtracking, while the polymerase alone exhibits low processivity and frequent exonuclease cleavage during strand-displacement synthesis [8,27,28]. Moreover, unwinding by an individual protein is passive and highly dependent on the base-pairing energy of parental DNA.…”