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. 2021 Jun 17;49(12):6804–6816. doi: 10.1093/nar/gkab493

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — DnaB helicases are both stable and dynamic during replication. (A) Illustration of the WT DnaB chase assay, where preloaded DnaB₆(red) was ‘chased’ with a relatively high concentration of WT DnaB (30 nM) in the replication solution. Like the standard rolling-circle assay, DNA products are stretched out by hydrodynamic force. (B) Representative kymograph of DnaB₆(red) moving with the fork during rolling-circle replication in the WT DnaB chase assay. (C) The average intensity over time from replicating DnaB₆(red) molecules in the WT DnaB chase assay (magenta; n = 29), compared to the photobleaching lifetime of DnaB₆(red) (grey; n = 667). The curve from each condition is fit with a single-exponential decay to provide the characteristic lifetime. (D) Illustration of the DnaB₆(blue) chase assay, where preloaded DnaB₆(red) is ‘chased’ with DnaB₆(blue) (2 nM) in the replication solution. Again, the DNA products are stretched out by hydrodynamic force. (E) (Top) Representative kymographs of DnaB₆(red) moving with the fork during rolling-circle replication in the DnaB₆(blue) chase assay. (Bottom) The DnaB₆(blue) kymograph from the same replication event shows the frequent association of additional helicases with the replication fork. (F) The stoichiometry over time from both the DnaB₆(red) and DnaB₆(blue) signal corresponding to the kymograph in (E), where steps are detected by change-point analysis (47–49).