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Rep provides a second motor at the replisome to promote duplication of protein-bound DNA.

Guy CP, Atkinson J, Gupta MK, Mahdi AA, Gwynn EJ, Rudolph CJ, Moon PB, van Knippenberg IC, Cadman CJ, Dillingham MS, Lloyd RG, McGlynn P - Mol. Cell (2009)

Bottom Line: However, these two helicases are not equivalent.Rep but not UvrD interacts physically and functionally with the replicative helicase.Rep and UvrD therefore provide two contrasting solutions as to how organisms may promote replication of protein-bound DNA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, UK.

ABSTRACT
Nucleoprotein complexes present challenges to genome stability by acting as potent blocks to replication. One attractive model of how such conflicts are resolved is direct targeting of blocked forks by helicases with the ability to displace the blocking protein-DNA complex. We show that Rep and UvrD each promote movement of E. coli replisomes blocked by nucleoprotein complexes in vitro, that such an activity is required to clear protein blocks (primarily transcription complexes) in vivo, and that a polarity of translocation opposite that of the replicative helicase is critical for this activity. However, these two helicases are not equivalent. Rep but not UvrD interacts physically and functionally with the replicative helicase. In contrast, UvrD likely provides a general means of protein-DNA complex turnover during replication, repair, and recombination. Rep and UvrD therefore provide two contrasting solutions as to how organisms may promote replication of protein-bound DNA.

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Related in: MedlinePlus

Rep and UvrD Promote Replication Fork Movement through EcoRI E111G-DNA Complexes In Vitro(A) Relative positions of oriC and EcoRI sites within plasmid templates, the site of cleavage by EagI, and the predicted sizes of leading strand products generated with and without replication blockage.(B) Denaturing agarose gel of replication products from pPM594 with and without E111G in the presence of the indicated helicases/translocases.(C) Levels of the 4.7 kb leading strand generated from pPM594 in the presence of E111G and the indicated enzymes relative to control reactions in lanes 1 and 2 in (B).(D) Replication products with pME101.(E) Levels of the 4.7 kb leading strand generated with pME101, plus E111G and the indicated enzymes relative to control reactions in lanes 1 and 2 in (D).(F) Relative levels of the 4.7 kb leading strand generated with pME101 plus E111G at increasing concentrations of Rep and UvrD. E111G was present at 200 nM dimers in all assays, while helicases/translocases were at 100 nM unless indicated otherwise. Error bars represent standard deviation of the mean.
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fig1: Rep and UvrD Promote Replication Fork Movement through EcoRI E111G-DNA Complexes In Vitro(A) Relative positions of oriC and EcoRI sites within plasmid templates, the site of cleavage by EagI, and the predicted sizes of leading strand products generated with and without replication blockage.(B) Denaturing agarose gel of replication products from pPM594 with and without E111G in the presence of the indicated helicases/translocases.(C) Levels of the 4.7 kb leading strand generated from pPM594 in the presence of E111G and the indicated enzymes relative to control reactions in lanes 1 and 2 in (B).(D) Replication products with pME101.(E) Levels of the 4.7 kb leading strand generated with pME101, plus E111G and the indicated enzymes relative to control reactions in lanes 1 and 2 in (D).(F) Relative levels of the 4.7 kb leading strand generated with pME101 plus E111G at increasing concentrations of Rep and UvrD. E111G was present at 200 nM dimers in all assays, while helicases/translocases were at 100 nM unless indicated otherwise. Error bars represent standard deviation of the mean.

Mentions: To search for helicases that might promote fork movement through protein-DNA complexes, we analyzed movement of reconstituted E. coli replisomes along template DNA bound by a model protein-DNA replication block. EcoRI E111G binds to its recognition sequence but has greatly reduced cleavage activity (King et al., 1989) (see also Figure S2B, lanes 1–3). Replication of plasmids bearing oriC and two or eight EcoRI sites was initiated by addition of DnaA and replisome components followed by cleavage with EagI. Cleavage enabled passage of a single fork through the EcoRI sites to be monitored, since fork progression in the absence of a topoisomerase could occur only after relief of replication-induced positive supercoiling by restriction enzyme cleavage (Marians et al., 1998) (Figures 1Ab and 1Ac). In the absence of E111G, replication generated lagging strands of approximately 0.5 kb and leading strands of 4.7 and 1.3 kb (Figures 1B and 1D, lane 1). Upon addition of E111G, there was a decrease in the amount of the 4.7 kb leading strand, together with the appearance of a 3.2 kb product with a greater inhibitory effect observed for eight as opposed to two EcoRI sites (compare lanes 1 and 2 in Figures 1B and 1D; Figure S1). This 3.2 kb product was the size expected if clockwise-moving forks stopped at the E111G complexes (Figure 1Ad). Generation of this 3.2 kb leading strand also required EcoRI sites within the template DNA in addition to E111G, while levels of this truncated leading strand were dependent on E111G concentration (Figure S1). EcoRI E111G bound to its cognate DNA-binding site therefore provided a barrier to replisome movement.


Rep provides a second motor at the replisome to promote duplication of protein-bound DNA.

Guy CP, Atkinson J, Gupta MK, Mahdi AA, Gwynn EJ, Rudolph CJ, Moon PB, van Knippenberg IC, Cadman CJ, Dillingham MS, Lloyd RG, McGlynn P - Mol. Cell (2009)

Rep and UvrD Promote Replication Fork Movement through EcoRI E111G-DNA Complexes In Vitro(A) Relative positions of oriC and EcoRI sites within plasmid templates, the site of cleavage by EagI, and the predicted sizes of leading strand products generated with and without replication blockage.(B) Denaturing agarose gel of replication products from pPM594 with and without E111G in the presence of the indicated helicases/translocases.(C) Levels of the 4.7 kb leading strand generated from pPM594 in the presence of E111G and the indicated enzymes relative to control reactions in lanes 1 and 2 in (B).(D) Replication products with pME101.(E) Levels of the 4.7 kb leading strand generated with pME101, plus E111G and the indicated enzymes relative to control reactions in lanes 1 and 2 in (D).(F) Relative levels of the 4.7 kb leading strand generated with pME101 plus E111G at increasing concentrations of Rep and UvrD. E111G was present at 200 nM dimers in all assays, while helicases/translocases were at 100 nM unless indicated otherwise. Error bars represent standard deviation of the mean.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2807033&req=5

fig1: Rep and UvrD Promote Replication Fork Movement through EcoRI E111G-DNA Complexes In Vitro(A) Relative positions of oriC and EcoRI sites within plasmid templates, the site of cleavage by EagI, and the predicted sizes of leading strand products generated with and without replication blockage.(B) Denaturing agarose gel of replication products from pPM594 with and without E111G in the presence of the indicated helicases/translocases.(C) Levels of the 4.7 kb leading strand generated from pPM594 in the presence of E111G and the indicated enzymes relative to control reactions in lanes 1 and 2 in (B).(D) Replication products with pME101.(E) Levels of the 4.7 kb leading strand generated with pME101, plus E111G and the indicated enzymes relative to control reactions in lanes 1 and 2 in (D).(F) Relative levels of the 4.7 kb leading strand generated with pME101 plus E111G at increasing concentrations of Rep and UvrD. E111G was present at 200 nM dimers in all assays, while helicases/translocases were at 100 nM unless indicated otherwise. Error bars represent standard deviation of the mean.
Mentions: To search for helicases that might promote fork movement through protein-DNA complexes, we analyzed movement of reconstituted E. coli replisomes along template DNA bound by a model protein-DNA replication block. EcoRI E111G binds to its recognition sequence but has greatly reduced cleavage activity (King et al., 1989) (see also Figure S2B, lanes 1–3). Replication of plasmids bearing oriC and two or eight EcoRI sites was initiated by addition of DnaA and replisome components followed by cleavage with EagI. Cleavage enabled passage of a single fork through the EcoRI sites to be monitored, since fork progression in the absence of a topoisomerase could occur only after relief of replication-induced positive supercoiling by restriction enzyme cleavage (Marians et al., 1998) (Figures 1Ab and 1Ac). In the absence of E111G, replication generated lagging strands of approximately 0.5 kb and leading strands of 4.7 and 1.3 kb (Figures 1B and 1D, lane 1). Upon addition of E111G, there was a decrease in the amount of the 4.7 kb leading strand, together with the appearance of a 3.2 kb product with a greater inhibitory effect observed for eight as opposed to two EcoRI sites (compare lanes 1 and 2 in Figures 1B and 1D; Figure S1). This 3.2 kb product was the size expected if clockwise-moving forks stopped at the E111G complexes (Figure 1Ad). Generation of this 3.2 kb leading strand also required EcoRI sites within the template DNA in addition to E111G, while levels of this truncated leading strand were dependent on E111G concentration (Figure S1). EcoRI E111G bound to its cognate DNA-binding site therefore provided a barrier to replisome movement.

Bottom Line: However, these two helicases are not equivalent.Rep but not UvrD interacts physically and functionally with the replicative helicase.Rep and UvrD therefore provide two contrasting solutions as to how organisms may promote replication of protein-bound DNA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, UK.

ABSTRACT
Nucleoprotein complexes present challenges to genome stability by acting as potent blocks to replication. One attractive model of how such conflicts are resolved is direct targeting of blocked forks by helicases with the ability to displace the blocking protein-DNA complex. We show that Rep and UvrD each promote movement of E. coli replisomes blocked by nucleoprotein complexes in vitro, that such an activity is required to clear protein blocks (primarily transcription complexes) in vivo, and that a polarity of translocation opposite that of the replicative helicase is critical for this activity. However, these two helicases are not equivalent. Rep but not UvrD interacts physically and functionally with the replicative helicase. In contrast, UvrD likely provides a general means of protein-DNA complex turnover during replication, repair, and recombination. Rep and UvrD therefore provide two contrasting solutions as to how organisms may promote replication of protein-bound DNA.

Show MeSH
Related in: MedlinePlus