DnaB Helicase Activity Is Modulated by DNA Geometry and Force

Abstract

The replicative helicase for Escherichia coli is DnaB, a hexameric, ring-shaped motor protein that encircles and translocates along ssDNA, unwinding dsDNA in advance of its motion. The microscopic mechanisms of DnaB are unknown; further, prior work has found that DnaB's activity is modified by other replication proteins, indicating some mechanistic flexibility. To investigate these issues, we quantified translocation and unwinding by single DnaB molecules in three tethered DNA geometries held under tension. Our data support the following conclusions: 1), Unwinding by DnaB is enhanced by force-induced destabilization of dsDNA. 2), The magnitude of this stimulation varies with the geometry of the tension applied to the DNA substrate, possibly due to interactions between the helicase and the occluded ssDNA strand. 3), DnaB unwinding and (to a lesser extent) translocation are interrupted by pauses, which are also dependent on force and DNA geometry. 4), DnaB moves slower when a large tension is applied to the helicase-bound strand, indicating that it must perform mechanical work to compact the strand against the applied force. Our results have implications for the molecular mechanisms of translocation and unwinding by DnaB and for the means of modulating DnaB activity.

Cdc45 Protein-Single-stranded DNA Interaction Is Important for Stalling the Helicase during Replication Stress

“…DnaB is highly processive and rapid in vitro without the addition of accessory factors (50,51). In contrast, the eukaryotic replication fork helicase requires the heterohexameric Mcm2-7 complex functioning in coordination with GINS and Cdc45 for processive and rapid unwinding (15,17).…”

Active DNA unwinding dynamics during processive DNA replication

“…2). The extension of the Betterton and Jülicher (11) model we present in this work points out the importance of considering pause kinetics and can be applied to the quantification of the unwinding mechanism of other NA unwinding motors in which pauses are known to be relevant or are expected to occur during their operation (17)(18)(19)(20).…”

“…Our data revealed that pauses strongly modulate the average unwinding rate of the mutant polymerase. Similarly, pauses have been shown also to be part of the unwinding mechanism of several specialized nucleic acid (NA) motors (17)(18)(19)(20). Therefore, the kinetics of pause events must be considered for the correct interpretation of tension and sequence dependencies of the unwinding rates.…”

Helicase and Polymerase Move Together Close to the Fork Junction and Copy DNA in One-Nucleotide Steps

“…In addition to DNA unwinding, replicative helicases play a key role in coordinating leading and lagging strand synthesis by mediating interactions with the polymerase and primase enzymes (Benkovic et al, 2001; Hamdan and Richardson, 2009; Indiani et al, 2009; Patel et al, 2011b). In isolation, replicative helicases are efficient single-stranded (ss)DNA translocases, but slow at unwinding the duplex DNA relying completely or partly on thermal fraying of the junction base pair (Donmez et al, 2007; Johnson et al, 2007; Kim et al, 2002; Lionnet et al, 2007; Ribeck et al, 2010) However, when the helicase is associated with the replicative polymerase, unwinding occurs at a fast rate (Dong et al, 1996; Kim et al., 1996; Korhonen et al, 2004; Manosas et al, 2012b; Stano et al, 2005). Similarly, replicative polymerases rely on helicases to processively copy the duplex DNA (Kang et al, 2012; Manosas et al, 2012b; Stano et al, 2005).…”