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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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Related in: MedlinePlus

Formation of stable Rep–DnaB–DNA complexes does not require branched DNA. (A) gel mobility shift assays with substrates 1–4 in the presence of 10 nM Rep and 10 nM DnaB hexamers as indicated. (B) gel mobility shift assays of substrates 1, 8 and 9 with 10 nM Rep and 100 nM DnaB hexamers. Note that similar patterns were also observed with substrates 1, 8 and 9 with 10 nM Rep and 10 nM DnaB hexamers (data not shown).
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Figure 6: Formation of stable Rep–DnaB–DNA complexes does not require branched DNA. (A) gel mobility shift assays with substrates 1–4 in the presence of 10 nM Rep and 10 nM DnaB hexamers as indicated. (B) gel mobility shift assays of substrates 1, 8 and 9 with 10 nM Rep and 100 nM DnaB hexamers. Note that similar patterns were also observed with substrates 1, 8 and 9 with 10 nM Rep and 10 nM DnaB hexamers (data not shown).

Mentions: Unwinding of a fork bearing a dsDNA lagging strand arm was also analysed. Unwinding by DnaB was inhibited as compared with substrates 1 and 2 (Figure 1A, compare lanes 3, 7 and 11), as expected given the requirement of this 5′-3′ translocase to bind to and translocate along the lagging strand arm. Unwinding of substrate 3 by Rep was also inhibited as compared with substrates 1 and 2 (Figure 1A, compare lanes 2, 6 and 10). The cause of this inhibition is unclear since translocation of Rep 3′–5′ along the leading strand arm should not have been inhibited by the duplex lagging strand arm. One possible explanation is that a higher binding affinity of Rep for the ssDNA on the lagging strand arm as compared with ssDNA on the leading strand arm (Figure 6A, compare lanes 6 and 10) might have increased the local concentration of Rep at the fork in substrate 1 as compared with 3, enhancing Rep-catalysed unwinding of substrate 1. However, this point was not explored further. Regardless of the cause of this reduced unwinding by Rep, the presence of both Rep and DnaB did not result in any enhancement of unwinding of this substrate (Figure 1A, lanes 10–12 and 1B). Similarly, no cooperativity of unwinding was observed when both leading and lagging strand arms were double-stranded (Figure 1A, lanes 13–16) as expected.


Interaction of Rep and DnaB on DNA.

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

Formation of stable Rep–DnaB–DNA complexes does not require branched DNA. (A) gel mobility shift assays with substrates 1–4 in the presence of 10 nM Rep and 10 nM DnaB hexamers as indicated. (B) gel mobility shift assays of substrates 1, 8 and 9 with 10 nM Rep and 100 nM DnaB hexamers. Note that similar patterns were also observed with substrates 1, 8 and 9 with 10 nM Rep and 10 nM DnaB hexamers (data not shown).
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Related In: Results  -  Collection

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Figure 6: Formation of stable Rep–DnaB–DNA complexes does not require branched DNA. (A) gel mobility shift assays with substrates 1–4 in the presence of 10 nM Rep and 10 nM DnaB hexamers as indicated. (B) gel mobility shift assays of substrates 1, 8 and 9 with 10 nM Rep and 100 nM DnaB hexamers. Note that similar patterns were also observed with substrates 1, 8 and 9 with 10 nM Rep and 10 nM DnaB hexamers (data not shown).
Mentions: Unwinding of a fork bearing a dsDNA lagging strand arm was also analysed. Unwinding by DnaB was inhibited as compared with substrates 1 and 2 (Figure 1A, compare lanes 3, 7 and 11), as expected given the requirement of this 5′-3′ translocase to bind to and translocate along the lagging strand arm. Unwinding of substrate 3 by Rep was also inhibited as compared with substrates 1 and 2 (Figure 1A, compare lanes 2, 6 and 10). The cause of this inhibition is unclear since translocation of Rep 3′–5′ along the leading strand arm should not have been inhibited by the duplex lagging strand arm. One possible explanation is that a higher binding affinity of Rep for the ssDNA on the lagging strand arm as compared with ssDNA on the leading strand arm (Figure 6A, compare lanes 6 and 10) might have increased the local concentration of Rep at the fork in substrate 1 as compared with 3, enhancing Rep-catalysed unwinding of substrate 1. However, this point was not explored further. Regardless of the cause of this reduced unwinding by Rep, the presence of both Rep and DnaB did not result in any enhancement of unwinding of this substrate (Figure 1A, lanes 10–12 and 1B). Similarly, no cooperativity of unwinding was observed when both leading and lagging strand arms were double-stranded (Figure 1A, lanes 13–16) as expected.

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