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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 between DnaB and Rep increases with the size of ssDNA exposed on the leading strand template at forks. Unwinding of substrates 2 and 5–7 was monitored in the presence of 10 nM Rep, 10 nM DnaB hexamers and 10 nM Rep +10 nM DnaB hexamers. The relative levels of unwinding by Rep plus DnaB in comparison to the sum of unwinding by each individual helicase is shown.
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Figure 3: Cooperativity between DnaB and Rep increases with the size of ssDNA exposed on the leading strand template at forks. Unwinding of substrates 2 and 5–7 was monitored in the presence of 10 nM Rep, 10 nM DnaB hexamers and 10 nM Rep +10 nM DnaB hexamers. The relative levels of unwinding by Rep plus DnaB in comparison to the sum of unwinding by each individual helicase is shown.

Mentions: Rep can promote movement of replisomes that have become blocked by nucleoprotein complexes but otherwise retain the ability to resume replication upon clearance of the block (14). A requirement for ssDNA to be present on the leading strand template implies therefore that such ssDNA is accessible even within the context of an active replisome. We probed therefore the length of ssDNA on the leading strand template required to observe cooperativity between Rep and DnaB. A series of substrates were constructed that differed only in the length of the ssDNA present on the leading strand template at the branch point of the fork. The degree of cooperativity increased as the length of this ssDNA on the leading strand template increased from 0 to 18 nt (Figure 3). Increased cooperativity was observed even with only 6 nt on the leading strand template. Given the 8-nt binding site size of Rep, as judged by X-ray crystallography (26), these data imply that initial access of even a single Rep monomer may be sufficient to facilitate cooperative unwinding of DNA in conjunction with DnaB.Figure 3.


Interaction of Rep and DnaB on DNA.

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

Cooperativity between DnaB and Rep increases with the size of ssDNA exposed on the leading strand template at forks. Unwinding of substrates 2 and 5–7 was monitored in the presence of 10 nM Rep, 10 nM DnaB hexamers and 10 nM Rep +10 nM DnaB hexamers. The relative levels of unwinding by Rep plus DnaB in comparison to the sum of unwinding by each individual helicase is shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Cooperativity between DnaB and Rep increases with the size of ssDNA exposed on the leading strand template at forks. Unwinding of substrates 2 and 5–7 was monitored in the presence of 10 nM Rep, 10 nM DnaB hexamers and 10 nM Rep +10 nM DnaB hexamers. The relative levels of unwinding by Rep plus DnaB in comparison to the sum of unwinding by each individual helicase is shown.
Mentions: Rep can promote movement of replisomes that have become blocked by nucleoprotein complexes but otherwise retain the ability to resume replication upon clearance of the block (14). A requirement for ssDNA to be present on the leading strand template implies therefore that such ssDNA is accessible even within the context of an active replisome. We probed therefore the length of ssDNA on the leading strand template required to observe cooperativity between Rep and DnaB. A series of substrates were constructed that differed only in the length of the ssDNA present on the leading strand template at the branch point of the fork. The degree of cooperativity increased as the length of this ssDNA on the leading strand template increased from 0 to 18 nt (Figure 3). Increased cooperativity was observed even with only 6 nt on the leading strand template. Given the 8-nt binding site size of Rep, as judged by X-ray crystallography (26), these data imply that initial access of even a single Rep monomer may be sufficient to facilitate cooperative unwinding of DNA in conjunction with DnaB.Figure 3.

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