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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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Cooperativity in unwinding by DnaB and Rep is observed on forked DNA bearing a ssDNA leading strand arm but not a dsDNA leading strand arm. (A) and (B) Unwinding of substrates 1 and 2 by the indicated concentrations of Rep in the absence and in the presence of 10 nM DnaB hexamers. (C) Relative levels of substrate unwinding by Rep plus DnaB in comparison to the sum of unwinding by each individual helicase.
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Figure 2: Cooperativity in unwinding by DnaB and Rep is observed on forked DNA bearing a ssDNA leading strand arm but not a dsDNA leading strand arm. (A) and (B) Unwinding of substrates 1 and 2 by the indicated concentrations of Rep in the absence and in the presence of 10 nM DnaB hexamers. (C) Relative levels of substrate unwinding by Rep plus DnaB in comparison to the sum of unwinding by each individual helicase.

Mentions: As shown previously (14), with a forked DNA possessing two ssDNA strands at the fork, cooperativity in unwinding was observed with Rep plus DnaB relative to the sum of unwinding by individual helicases (Figure 1A, lanes 1–4; Figure 2A and C). Moreover, cooperativity was observed on this substrate regardless of the order of addition of Rep and DnaB (data not shown). The relative importance of ssDNA being present on the arm equivalent to the leading strand template was tested using substrate 2, bearing dsDNA rather than ssDNA on this arm. Unwinding of this substrate by Rep was inhibited as compared with substrate 1 (Figure 1A, compare lanes 2 and 6 and 1B), reflecting a requirement for this 3′–5′ ssDNA translocase to bind to and move along the leading strand template to effect unwinding of such substrates. In contrast, unwinding by DnaB alone resulted in a greater fraction of substrate 2 being unwound as compared with substrate 1 (Figure 1A, compare lanes 3 and 7 and 1B). Such stimulation by the presence of dsDNA as opposed to ssDNA on the leading strand arm is consistent with previous observations (25). This stimulation is likely due to the increased rigidity of this dsDNA arm reducing the probability of this arm being located within the central cavity of DnaB as it translocates along the lagging strand arm, a reaction that would not result in unwinding of the substrate (25). Upon addition of both Rep and DnaB to substrate 2, no major enhancement of unwinding was observed as compared with each individual helicase (Figure 1A, compare lanes 6–8 and 1B). This lack of enhancement of unwinding of substrate 2 was observed at all tested concentrations of Rep (Figure 2B and C). The single-stranded versus double-stranded nature of the leading strand arm has therefore a major impact on cooperative unwinding of branched DNA by Rep and DnaB.Figure 1.


Interaction of Rep and DnaB on DNA.

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

Cooperativity in unwinding by DnaB and Rep is observed on forked DNA bearing a ssDNA leading strand arm but not a dsDNA leading strand arm. (A) and (B) Unwinding of substrates 1 and 2 by the indicated concentrations of Rep in the absence and in the presence of 10 nM DnaB hexamers. (C) Relative levels of substrate unwinding by Rep plus DnaB in comparison to the sum of unwinding by each individual helicase.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3045612&req=5

Figure 2: Cooperativity in unwinding by DnaB and Rep is observed on forked DNA bearing a ssDNA leading strand arm but not a dsDNA leading strand arm. (A) and (B) Unwinding of substrates 1 and 2 by the indicated concentrations of Rep in the absence and in the presence of 10 nM DnaB hexamers. (C) Relative levels of substrate unwinding by Rep plus DnaB in comparison to the sum of unwinding by each individual helicase.
Mentions: As shown previously (14), with a forked DNA possessing two ssDNA strands at the fork, cooperativity in unwinding was observed with Rep plus DnaB relative to the sum of unwinding by individual helicases (Figure 1A, lanes 1–4; Figure 2A and C). Moreover, cooperativity was observed on this substrate regardless of the order of addition of Rep and DnaB (data not shown). The relative importance of ssDNA being present on the arm equivalent to the leading strand template was tested using substrate 2, bearing dsDNA rather than ssDNA on this arm. Unwinding of this substrate by Rep was inhibited as compared with substrate 1 (Figure 1A, compare lanes 2 and 6 and 1B), reflecting a requirement for this 3′–5′ ssDNA translocase to bind to and move along the leading strand template to effect unwinding of such substrates. In contrast, unwinding by DnaB alone resulted in a greater fraction of substrate 2 being unwound as compared with substrate 1 (Figure 1A, compare lanes 3 and 7 and 1B). Such stimulation by the presence of dsDNA as opposed to ssDNA on the leading strand arm is consistent with previous observations (25). This stimulation is likely due to the increased rigidity of this dsDNA arm reducing the probability of this arm being located within the central cavity of DnaB as it translocates along the lagging strand arm, a reaction that would not result in unwinding of the substrate (25). Upon addition of both Rep and DnaB to substrate 2, no major enhancement of unwinding was observed as compared with each individual helicase (Figure 1A, compare lanes 6–8 and 1B). This lack of enhancement of unwinding of substrate 2 was observed at all tested concentrations of Rep (Figure 2B and C). The single-stranded versus double-stranded nature of the leading strand arm has therefore a major impact on cooperative unwinding of branched DNA by Rep and DnaB.Figure 1.

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