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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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Correlation between Helicase-Mediated Promotion of Fork Movement In Vitro and Complementation of Δrep ΔuvrD Lethality In Vivo(A) Denaturing agarose gel of replication products from pME101 (two EcoRI sites) with and without E111G (200 nM dimers) in the presence of the indicated helicases (100 nM).(B) Levels of the 4.7 kb leading strand in the presence of the indicated helicases relative to control reactions in lanes 2 and 3 in (A). Error bars represent standard deviation of the mean.(C) Scheme for generation of strains containing helicase genes under the control of an arabinose-inducible promoter.(D and E) Colony-forming ability of rep+uvrD+ (N6524) and Δrep ΔuvrD (N6556) strains lacking pRC7rep but bearing the indicated plasmids after growth in liquid minimal medium, subsequent dilution, and spotting onto LB containing kanamycin ± arabinose.
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fig5: Correlation between Helicase-Mediated Promotion of Fork Movement In Vitro and Complementation of Δrep ΔuvrD Lethality In Vivo(A) Denaturing agarose gel of replication products from pME101 (two EcoRI sites) with and without E111G (200 nM dimers) in the presence of the indicated helicases (100 nM).(B) Levels of the 4.7 kb leading strand in the presence of the indicated helicases relative to control reactions in lanes 2 and 3 in (A). Error bars represent standard deviation of the mean.(C) Scheme for generation of strains containing helicase genes under the control of an arabinose-inducible promoter.(D and E) Colony-forming ability of rep+uvrD+ (N6524) and Δrep ΔuvrD (N6556) strains lacking pRC7rep but bearing the indicated plasmids after growth in liquid minimal medium, subsequent dilution, and spotting onto LB containing kanamycin ± arabinose.

Mentions: Δrep ΔuvrD lethality can be complemented by a Rep/UvrD homolog from Bacillus, PcrA (Petit et al., 1998). We found that PcrA, like Rep and UvrD, promoted movement of reconstituted E. coli replisomes through DNA-E111G complexes (Figures 5A and 5B). Rep, UvrD, and PcrA are all Superfamily 1 helicases that translocate 3′-5′ along ssDNA to effect duplex unwinding (Singleton et al., 2007). We tested whether Superfamily 1 helicases that move in the opposite direction along ssDNA also promote replisome movement along protein-bound DNA. However, neither bacteriophage T4 Dda (Jongeneel et al., 1984) nor Deinococcus radiodurans RecD2 (Wang and Julin, 2004) could promote movement of E. coli replisomes through E111G complexes in vitro (Figures 5A and 5B). Indeed, addition of RecD2 resulted in decreased production of the full-length 4.7 kb leading strand (Figure 5B), implying that RecD2 reduced readthrough of replisomes at DNA-E111G complexes. Thus, the ability of helicases to promote fork movement through protein-DNA complexes in vitro correlates with polarity of helicase translocation along ssDNA. This translocation polarity, 3′-5′, is opposite to that of the replicative helicase DnaB.


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)

Correlation between Helicase-Mediated Promotion of Fork Movement In Vitro and Complementation of Δrep ΔuvrD Lethality In Vivo(A) Denaturing agarose gel of replication products from pME101 (two EcoRI sites) with and without E111G (200 nM dimers) in the presence of the indicated helicases (100 nM).(B) Levels of the 4.7 kb leading strand in the presence of the indicated helicases relative to control reactions in lanes 2 and 3 in (A). Error bars represent standard deviation of the mean.(C) Scheme for generation of strains containing helicase genes under the control of an arabinose-inducible promoter.(D and E) Colony-forming ability of rep+uvrD+ (N6524) and Δrep ΔuvrD (N6556) strains lacking pRC7rep but bearing the indicated plasmids after growth in liquid minimal medium, subsequent dilution, and spotting onto LB containing kanamycin ± arabinose.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Correlation between Helicase-Mediated Promotion of Fork Movement In Vitro and Complementation of Δrep ΔuvrD Lethality In Vivo(A) Denaturing agarose gel of replication products from pME101 (two EcoRI sites) with and without E111G (200 nM dimers) in the presence of the indicated helicases (100 nM).(B) Levels of the 4.7 kb leading strand in the presence of the indicated helicases relative to control reactions in lanes 2 and 3 in (A). Error bars represent standard deviation of the mean.(C) Scheme for generation of strains containing helicase genes under the control of an arabinose-inducible promoter.(D and E) Colony-forming ability of rep+uvrD+ (N6524) and Δrep ΔuvrD (N6556) strains lacking pRC7rep but bearing the indicated plasmids after growth in liquid minimal medium, subsequent dilution, and spotting onto LB containing kanamycin ± arabinose.
Mentions: Δrep ΔuvrD lethality can be complemented by a Rep/UvrD homolog from Bacillus, PcrA (Petit et al., 1998). We found that PcrA, like Rep and UvrD, promoted movement of reconstituted E. coli replisomes through DNA-E111G complexes (Figures 5A and 5B). Rep, UvrD, and PcrA are all Superfamily 1 helicases that translocate 3′-5′ along ssDNA to effect duplex unwinding (Singleton et al., 2007). We tested whether Superfamily 1 helicases that move in the opposite direction along ssDNA also promote replisome movement along protein-bound DNA. However, neither bacteriophage T4 Dda (Jongeneel et al., 1984) nor Deinococcus radiodurans RecD2 (Wang and Julin, 2004) could promote movement of E. coli replisomes through E111G complexes in vitro (Figures 5A and 5B). Indeed, addition of RecD2 resulted in decreased production of the full-length 4.7 kb leading strand (Figure 5B), implying that RecD2 reduced readthrough of replisomes at DNA-E111G complexes. Thus, the ability of helicases to promote fork movement through protein-DNA complexes in vitro correlates with polarity of helicase translocation along ssDNA. This translocation polarity, 3′-5′, is opposite to that of the replicative helicase DnaB.

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