Caracterización del mecanismo de acción de la helicasa replicativa G40P y su cargador G39P en la replicación del bacteriófago SPP1 de Bacillus subtilis

Resumen

The Bacillus subtilis SPP1-encoded G40P is a hexameric replicative helicase that unwinds dsDNA with a 5¿!3¿ polarity. During the amplification of the SPP1 genome this DnaB-like helicase uses its ATP-powered motor activity to provide ssDNA to the host polymerases, the DnaG primase and the DNA polymerases (polC and DnaE). G40P is also involved in the initiation of SPP1 DNA replication, with two other phage-encoded proteins: G39P and G38P. Through its interactions with G40P and G38P, G39P is able to load the helicase into the replication origin (oriL), which is recognized and locally unwound by G38P. This system represents an alternative mechanism to the reference model exemplified by Escherichia coli DNA replication initiation proteins: DnaA, DnaC and DnaB. This work addresses the study of G40P helicase activity and the role of G39P in the helicase loading mechanism. Different deletion constructs were used in order to link functions to structural features of these two proteins. In this regard, a G40P mutant lacking the N-terminal domain, G40P¿N109, showed two unexpected activities. It was able to unwinds DNA bidirectionally, with an additional 3¿!5¿ translocation activity which is absent in wild-type G40P. Surprisingly, G40P¿N109 was also capable of anneal complementary DNA strands. In order to explain these activities, we propose a regulatory function for the N-terminal domain of G40P. This part of the helicase could be involved in the control of the relative disposition of the ATPase C-terminal domains and in the regulation of the DNA-binding activity. Structural and biochemical studies showed that G39P contains two different domains, a folded N-terminal domain and a natively disordered C-terminal domain. We propose that this bimodular structure is essential for G39P to function as a helicase loader. The interaction of the disordered regions with G40P would induce the association of the N-terminal domains of G39P, and this event could force the opening of the helicase ring. Then G40P is loaded on the DNA and G39P is released. By using this mechanism G39P promotes the loading of G40P on ssDNA and prevents the loading on dsDNA. In addition, small natural compounds griseorhodin C and purpuromycin were found to inhibit the ATPase and helicase activities of G40P.