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Interaction of Rep and DnaB on DNA.

Atkinson J, Gupta MK, McGlynn P - Nucleic Acids Res. (2010)

Bottom Line: However, accessory helicases are also needed since the replicative helicase stalls occasionally at nucleoprotein complexes.In Escherichia coli, the primary and accessory helicases DnaB and Rep translocate along the lagging and leading strand templates, respectively, interact physically and also display cooperativity in the unwinding of model forked DNA substrates.However, stable Rep-DnaB complexes can form on linear as well as branched DNA, indicating that Rep has the capacity to interact with ssDNA on either the leading or the lagging strand template at forks.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.

ABSTRACT
Genome duplication requires not only unwinding of the template but also the displacement of proteins bound to the template, a function performed by replicative helicases located at the fork. However, accessory helicases are also needed since the replicative helicase stalls occasionally at nucleoprotein complexes. In Escherichia coli, the primary and accessory helicases DnaB and Rep translocate along the lagging and leading strand templates, respectively, interact physically and also display cooperativity in the unwinding of model forked DNA substrates. We demonstrate here that this cooperativity is displayed only by Rep and not by other tested helicases. ssDNA must be exposed on the leading strand template to elicit this cooperativity, indicating that forks blocked at protein-DNA complexes contain ssDNA ahead of the leading strand polymerase. However, stable Rep-DnaB complexes can form on linear as well as branched DNA, indicating that Rep has the capacity to interact with ssDNA on either the leading or the lagging strand template at forks. Inhibition of Rep binding to the lagging strand template by competition with SSB might therefore be critical in targeting accessory helicases to the leading strand template, indicating an important role for replisome architecture in promoting accessory helicase function at blocked replisomes.

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Binding of Rep is not sufficient to enhance DNA unwinding by DnaB. (A) Unwinding of substrate 1 by the indicated concentrations of wild-type Rep in the absence and in the presence of 10 nM DnaB hexamers. (B) Unwinding of substrate 1 by RepK28A without and with 10 nM DnaB hexamers.
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Figure 4: Binding of Rep is not sufficient to enhance DNA unwinding by DnaB. (A) Unwinding of substrate 1 by the indicated concentrations of wild-type Rep in the absence and in the presence of 10 nM DnaB hexamers. (B) Unwinding of substrate 1 by RepK28A without and with 10 nM DnaB hexamers.

Mentions: One model to explain the observed cooperativity between Rep and DnaB is that physical coupling of two active helicases at a fork increases the probability of substrate unwinding. Alternatively, binding of one motor to the fork might act merely as a platform for the second motor, increasing the local concentration of the second motor at the fork. We tested this possibility by using a mutant Rep in which the invariant lysine within helicase motif I, known to be essential for ATP hydrolysis and hence helicase activity (26–30), was replaced by alanine. Absence of cooperativity between RepK28A and DnaB indicated that Rep helicase activity was essential for enhancement of unwinding with DnaB (Figure 4). This lack of cooperativity was reflected in the inability of RepK28A to promote E. coli replication fork movement through protein–DNA complexes in vitro (data not shown). We were unable to test whether binding of DnaB was sufficient to stimulate Rep-catalysed unwinding since mutation of the equivalent helicase motif I lysine in DnaB rendered the protein insoluble (data not shown). However, these data do indicate that Rep does not play a merely structural role in stimulation of DnaB and promotion of replisome movement along protein-bound DNA.Figure 4.


Interaction of Rep and DnaB on DNA.

Atkinson J, Gupta MK, McGlynn P - Nucleic Acids Res. (2010)

Binding of Rep is not sufficient to enhance DNA unwinding by DnaB. (A) Unwinding of substrate 1 by the indicated concentrations of wild-type Rep in the absence and in the presence of 10 nM DnaB hexamers. (B) Unwinding of substrate 1 by RepK28A without and with 10 nM DnaB hexamers.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Binding of Rep is not sufficient to enhance DNA unwinding by DnaB. (A) Unwinding of substrate 1 by the indicated concentrations of wild-type Rep in the absence and in the presence of 10 nM DnaB hexamers. (B) Unwinding of substrate 1 by RepK28A without and with 10 nM DnaB hexamers.
Mentions: One model to explain the observed cooperativity between Rep and DnaB is that physical coupling of two active helicases at a fork increases the probability of substrate unwinding. Alternatively, binding of one motor to the fork might act merely as a platform for the second motor, increasing the local concentration of the second motor at the fork. We tested this possibility by using a mutant Rep in which the invariant lysine within helicase motif I, known to be essential for ATP hydrolysis and hence helicase activity (26–30), was replaced by alanine. Absence of cooperativity between RepK28A and DnaB indicated that Rep helicase activity was essential for enhancement of unwinding with DnaB (Figure 4). This lack of cooperativity was reflected in the inability of RepK28A to promote E. coli replication fork movement through protein–DNA complexes in vitro (data not shown). We were unable to test whether binding of DnaB was sufficient to stimulate Rep-catalysed unwinding since mutation of the equivalent helicase motif I lysine in DnaB rendered the protein insoluble (data not shown). However, these data do indicate that Rep does not play a merely structural role in stimulation of DnaB and promotion of replisome movement along protein-bound DNA.Figure 4.

Bottom Line: However, accessory helicases are also needed since the replicative helicase stalls occasionally at nucleoprotein complexes.In Escherichia coli, the primary and accessory helicases DnaB and Rep translocate along the lagging and leading strand templates, respectively, interact physically and also display cooperativity in the unwinding of model forked DNA substrates.However, stable Rep-DnaB complexes can form on linear as well as branched DNA, indicating that Rep has the capacity to interact with ssDNA on either the leading or the lagging strand template at forks.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.

ABSTRACT
Genome duplication requires not only unwinding of the template but also the displacement of proteins bound to the template, a function performed by replicative helicases located at the fork. However, accessory helicases are also needed since the replicative helicase stalls occasionally at nucleoprotein complexes. In Escherichia coli, the primary and accessory helicases DnaB and Rep translocate along the lagging and leading strand templates, respectively, interact physically and also display cooperativity in the unwinding of model forked DNA substrates. We demonstrate here that this cooperativity is displayed only by Rep and not by other tested helicases. ssDNA must be exposed on the leading strand template to elicit this cooperativity, indicating that forks blocked at protein-DNA complexes contain ssDNA ahead of the leading strand polymerase. However, stable Rep-DnaB complexes can form on linear as well as branched DNA, indicating that Rep has the capacity to interact with ssDNA on either the leading or the lagging strand template at forks. Inhibition of Rep binding to the lagging strand template by competition with SSB might therefore be critical in targeting accessory helicases to the leading strand template, indicating an important role for replisome architecture in promoting accessory helicase function at blocked replisomes.

Show MeSH
Related in: MedlinePlus