The DnaC helicase loader is a dual ATP/ADP switch protein

Abstract

Helicases are transferred to replication origins by helicase loading factors. The Escherichia coli DnaC and eukaryotic Cdc6/18 helicase loaders contain ATP sites and are both members of the AAA+ family. One might expect that ATP is required for helicase loading; however, this study on DnaC illustrates that ATP is not actually needed for DnaC to load helicase onto single‐strand DNA (ssDNA). In fact, it seems to be a paradox that after transfer of helicase to DNA, DnaC–ATP inhibits helicase action. In addition, ATP is required for DnaC function at an early step in oriC replication in which ATP stimulates ssDNA binding by DnaC, leading to expansion of the ssDNA bubble at the origin. Two cofactors, ssDNA and DnaB, trigger hydrolysis of ATP, converting DnaC to the ADP form that no longer inhibits DnaB. These observations have led to the idea that DnaC is a ‘dual’ switch protein, where both the ATP and the ADP forms are sequentially required for replication. This dual switching process may underlie the sensitivity of DnaB to even small fluctuations in DnaC levels.

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

“…Like the noncanonical variants of ClpB described in this work, DnaC is not an efficient ATPase, but unlike ClpB, it does not form oligomers. 18 Single-stranded DNA and the helicase DnaB (which is a canonical AAAþ ATPase) stimulate the ATP hydrolysis by DnaC. Moreover, DnaC assembles into oligomers on a scaffold of the hexameric DnaB.…”

“…Besides the torsin family, an Asn in the Walker A motif is found in DnaC, a bacterial AAAþ helicase loader. 18 Intriguingly, the biochemical properties of torsinA and DnaC do not fully conform to the AAAþ paradigm: those proteins do not form hexamers and have low ATPase activity in the absence of other macromolecules. 18,19 We hypothesized that the last residue of the Walker A motif may participate in coupling the binding of nucleotides with interactions of an AAAþ ATPase with substrates or partner proteins.…”

“…18 Intriguingly, the biochemical properties of torsinA and DnaC do not fully conform to the AAAþ paradigm: those proteins do not form hexamers and have low ATPase activity in the absence of other macromolecules. 18,19 We hypothesized that the last residue of the Walker A motif may participate in coupling the binding of nucleotides with interactions of an AAAþ ATPase with substrates or partner proteins. We replaced the Walker-A Thr in each AAAþ module of ClpB with Asn and discovered that the noncanonical sequence partially inhibited the ATPase and aggregatereactivation activity, modified the nucleotide-binding affinity, and altered the linkage between nucleotide-occupancy and aggregate binding.…”

Helicase Loading at Chromosomal Origins of Replication

“…Early studies suggested that ATP bound to DnaC is necessary for DnaC to form an isolable complex with DnaB and to protect DnaC from inactivation by N-ethylmaleimide (Wickner and Hurwitz 1975;Kobori and Kornberg 1982). More recent studies indicate that ATP is not needed for DnaC to interact with DnaB (Davey et al 2002;Galletto et al 2003;Mott et al 2008). Both cryoelectron microscopy of the DnaB -DnaC complex in comparison with DnaB and fluorescence energy transfer experiments indicate that DnaC is bound to the larger (carboxy-terminal) end of the DnaB toroid (Bárcena et al 2001;Galletto et al 2003).…”

“…Despite the presence in DnaC of motifs shared by AAA þ family members and the high affinity of other AAA þ proteins for ATP, DnaC binds weakly to this nucleotide; the K d is about 8 mM (Davey et al 2002;Galletto et al 2003;Biswas et al 2004). By itself, DnaC is also a feeble ATPase.…”