Direct removal of RNA polymerase barriers to replication by accessory replicative helicases.

Jamieson A L Howard,Nigel J Savery,Michelle Hawkins,Juachi U Dimude,Mark S Dillingham,Peter Mcglynn,Abigail J Smith,Christian J Rudolph

doi:10.1093/nar/gkz170

Jamieson A L Howard, Nigel J Savery + Show 6 more

Open Access

https://doi.org/10.1093/nar/gkz170

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Abstract

Bacterial genome duplication and transcription require simultaneous access to the same DNA template. Conflicts between the replisome and transcription machinery can lead to interruption of DNA replication and loss of genome stability. Pausing, stalling and backtracking of transcribing RNA polymerases add to this problem and present barriers to replisomes. Accessory helicases promote fork movement through nucleoprotein barriers and exist in viruses, bacteria and eukaryotes. Here, we show that stalled Escherichia coli transcription elongation complexes block reconstituted replisomes. This physiologically relevant block can be alleviated by the accessory helicase Rep or UvrD, resulting in the formation of full-length replication products. Accessory helicase action during replication-transcription collisions therefore promotes continued replication without leaving gaps in the DNA. In contrast, DinG does not promote replisome movement through stalled transcription complexes in vitro. However, our data demonstrate that DinG operates indirectly in vivo to reduce conflicts between replication and transcription. These results suggest that Rep and UvrD helicases operate on DNA at the replication fork whereas DinG helicase acts via a different mechanism.

Highlights

Synthetic lethality assay The synthetic lethality assay was performed as described (Bernhardt and de Boer 2003 PMID 12787347; (5)
Additional mutations can be introduced to test for synthetic lethality with the deleted allele
Cultures of strains carrying the relevant pRC7 derivatives were grown overnight in LB broth containing ampicillin to maintain plasmid selection, diluted 100-fold in LB broth and grown without ampicillin selection to an A600 of 0.4 before spreading dilutions on LB agar or M9 glucose minimal salts agar supplemented with X-gal and IPTG

Summary

Introduction

Synthetic lethality assay The synthetic lethality assay was performed as described (Bernhardt and de Boer 2003 PMID 12787347; (5). RNA polymerase stalled at PlacUV5 52C is a stable block. RNA polymerase was added to pPM872 with ATP, GTP and UTP but in the absence of CTP. The formation of linearised plasmid indicated occlusion of the NcoI site by stalled RNA polymerase.

Results

Conclusion