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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

Suppressed Δrep ΔuvrD Cells Remain Hypersensitive to an Artificial Nucleoprotein Barrier to Replication(A–D) Strains PM462–465 bearing 34 chromosomal lac operators plus pPM306, a plasmid bearing lacI under the control of an arabinose-inducible promoter, were tested for colony-forming ability upon expression of lac repressor. Cells were grown in LB in the absence of arabinose, and then serial dilutions were spotted onto LB agar containing arabinose without and with IPTG.
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fig4: Suppressed Δrep ΔuvrD Cells Remain Hypersensitive to an Artificial Nucleoprotein Barrier to Replication(A–D) Strains PM462–465 bearing 34 chromosomal lac operators plus pPM306, a plasmid bearing lacI under the control of an arabinose-inducible promoter, were tested for colony-forming ability upon expression of lac repressor. Cells were grown in LB in the absence of arabinose, and then serial dilutions were spotted onto LB agar containing arabinose without and with IPTG.

Mentions: Thirty-four chromosomal tandem lac repressor-operator complexes provide an inducible replicative barrier that is tolerated in otherwise WT cells and in cells lacking either Rep or UvrD (Payne et al., 2006). lacO34 was introduced into rpoB∗35 strains, and the consequences of repressor expression on colony-forming ability were evaluated in the presence or absence of isopropyl-β-D-thiogalactopyranoside (IPTG). Expression of repressor had no detectable effect on viability of rep+uvr+ rpoB∗35 or of strains bearing mutations in either helicase gene with or without IPTG (Figures 4A–4C). However, the colony-forming ability of Δrep ΔuvrD rpoB∗35 was reduced dramatically upon repressor expression, a reduction that was alleviated by IPTG (Figure 4D). Suppression by rpoB∗35 was reversed, therefore, by the tandem repressor-operator complexes, demonstrating that cells lacking both Rep and UvrD have a reduced inherent capacity to tolerate stable nucleoprotein complexes. Thus, suppression by rpoB∗35 does not occur via altered patterns of gene expression, nor by circumvention of direct roles of Rep or UvrD in transcription. Instead, these data strongly support a model in which rpoB∗35 suppresses Δrep ΔuvrD lethality by direct reduction of the replicative barriers posed by transcription complexes.


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)

Suppressed Δrep ΔuvrD Cells Remain Hypersensitive to an Artificial Nucleoprotein Barrier to Replication(A–D) Strains PM462–465 bearing 34 chromosomal lac operators plus pPM306, a plasmid bearing lacI under the control of an arabinose-inducible promoter, were tested for colony-forming ability upon expression of lac repressor. Cells were grown in LB in the absence of arabinose, and then serial dilutions were spotted onto LB agar containing arabinose without and with IPTG.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Suppressed Δrep ΔuvrD Cells Remain Hypersensitive to an Artificial Nucleoprotein Barrier to Replication(A–D) Strains PM462–465 bearing 34 chromosomal lac operators plus pPM306, a plasmid bearing lacI under the control of an arabinose-inducible promoter, were tested for colony-forming ability upon expression of lac repressor. Cells were grown in LB in the absence of arabinose, and then serial dilutions were spotted onto LB agar containing arabinose without and with IPTG.
Mentions: Thirty-four chromosomal tandem lac repressor-operator complexes provide an inducible replicative barrier that is tolerated in otherwise WT cells and in cells lacking either Rep or UvrD (Payne et al., 2006). lacO34 was introduced into rpoB∗35 strains, and the consequences of repressor expression on colony-forming ability were evaluated in the presence or absence of isopropyl-β-D-thiogalactopyranoside (IPTG). Expression of repressor had no detectable effect on viability of rep+uvr+ rpoB∗35 or of strains bearing mutations in either helicase gene with or without IPTG (Figures 4A–4C). However, the colony-forming ability of Δrep ΔuvrD rpoB∗35 was reduced dramatically upon repressor expression, a reduction that was alleviated by IPTG (Figure 4D). Suppression by rpoB∗35 was reversed, therefore, by the tandem repressor-operator complexes, demonstrating that cells lacking both Rep and UvrD have a reduced inherent capacity to tolerate stable nucleoprotein complexes. Thus, suppression by rpoB∗35 does not occur via altered patterns of gene expression, nor by circumvention of direct roles of Rep or UvrD in transcription. Instead, these data strongly support a model in which rpoB∗35 suppresses Δrep ΔuvrD lethality by direct reduction of the replicative barriers posed by transcription complexes.

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