ABSTRACT
Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD) of H. pylori DnaB (HpDnaB) helicase at 2.2 A resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.
Subject(s)
DNA Primase/chemistry , DnaB Helicases/chemistry , Helicobacter pylori/enzymology , Cloning, Molecular , Cohort Studies , Crystallography, X-Ray/methods , DNA/chemistry , Dimerization , Escherichia coli/metabolism , Helicobacter pylori/metabolism , Hydrolysis , Molecular Conformation , Protein Structure, Tertiary , Static Electricity , Surface Plasmon ResonanceABSTRACT
DNA replication takes place at five different stages during the life cycle of Plasmodium falciparum including the human and mosquito hosts. DNA replication initiation, the rate-determining step is poorly understood in Plasmodium. Here we show that PfMCM4 and PfORC1, two members of prereplication initiation complex are expressed specifically in the nucleus during the trophozoite and schizont stages of the asexual parasitic life cycle where maximum amount of DNA replication takes place. Further, we show that these proteins are also expressed in gametocytes, where DNA replication also occurs. These results expand our knowledge on these proteins and resolves discrepancies arising from previous studies with respect to the expression pattern of replication initiation proteins during the parasite's life cycle.
