Abstract
Replisome assembly requires the loading of replicative hexameric helicases onto origins by AAA+ ATPases. How loader activity is appropriately controlled remains unclear. Here, we use structural and biochemical analyses to establish how an antimicrobial phage protein interferes with the function of the Staphylococcus aureus replicative helicase loader, DnaI. The viral protein binds to the loader's AAA+ ATPase domain, allowing binding of the host replicative helicase but impeding loader self-assembly and ATPase activity. Close inspection of the complex highlights an unexpected locus for the binding of an interdomain linker element in DnaI/DnaC-family proteins. We find that the inhibitor protein is genetically coupled to a phage-encoded homolog of the bacterial helicase loader, which we show binds to the host helicase but not to the inhibitor itself. These findings establish a new approach by which viruses can hijack host replication processes and explain how loader activity is internally regulated to prevent aberrant auto-association.
Highlights
All cells face the challenging task of copying and passing on genetic information to progeny in an error-free manner as possible (Fuchs and Fujii, 2013 ; Sutera and Lovett, 2006)
Specific helicase loading mechanisms vary across the three domains of life – archaea, bacteria, and eukaryotes – all appear to rely on replication initiation factors belonging to the AAA+ (ATPases Associated with various cellular Activities) superfamily of nucleotide hydrolases
Turning again to pull-down assays with tagged 77ORF104, we found that the phage protein was capable of retaining both the isolated C-terminal AAA+ ATPase domain but not the N-terminal domain on its own (Figure 2A–B)
Summary
All cells face the challenging task of copying and passing on genetic information to progeny in an error-free manner as possible (Fuchs and Fujii, 2013 ; Sutera and Lovett, 2006). Replication initiation relies on the DnaA initiator, which recognizes and marks the bacterial replication origin (Bramhill and Kornberg, 1988a; 1988b; Funnell et al, 1987; Hsu et al, 1994). DnaA actively opens an AT-rich region of the origin (Bramhill and Kornberg, 1988a, 1988b; Dixon and Kornberg, 1984; Funnell et al, 1987; Gille and Messer, 1991; Hsu et al, 1994; Skarstad et al, 1990), termed a DNA-unwinding element (DUE) (Kowalski and Eddy, 1989), and helps to recruit two copies of the replicative helicase to the newly melted single strands. In certain Gram-negative bacteria, a protein known as DnaC assists with loading of the helicase (known in these organisms as DnaB); many Gram-
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