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Modulation of DNA repair by mutations flanking the DNA channel through RNA polymerase
Brigitte W Trautinger1, Robert G Lloyd
1Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK.
Abstract:
The RuvABC and RecBCD proteins promote rescue of stalled or broken DNA replication forks in Escherichia coli. Strains lacking these proteins cope poorly with DNA damage and have problems with chromosome segregation and cell division. We show how these difficulties are overcome to varying degrees by a sub-class of RNA polymerase mutations selected for their stringent phenotype. Thirty-five mutations were sequenced. All but one change single amino acids in RpoB or RpoC that lie on or near the path taken by DNA through the enzyme, indicating they may affect the stability of transcription complexes. Four mutant enzymes are shown to form unstable open complexes at the lambdacro promoter. At least one may also release stalled complexes or limit their formation, as it reduces the need for reactivation of transcription by GreA or GreB, and for transcription-coupled DNA repair of UV damage by Mfd. The results shed light on the interplay between DNA replication and transcription and suggest ways in which conflicts between these two vital cellular processes are avoided or resolved.
Insights
RNA polymerase mutations help Escherichia coli overcome DNA replication and repair challenges. These mutations stabilize transcription complexes, aiding cell survival when DNA repair proteins are absent.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- DNA replication forks can stall or break, requiring proteins like RuvABC and RecBCD for repair in Escherichia coli.
- Deficiencies in these DNA repair proteins lead to poor DNA damage tolerance, chromosome segregation, and cell division issues.
Purpose of the Study:
- To investigate how specific RNA polymerase mutations compensate for the lack of essential DNA repair proteins.
- To understand the molecular mechanisms by which these mutations improve cellular tolerance to DNA damage.
Main Methods:
- Sequencing of 35 RNA polymerase mutations conferring a stringent phenotype.
- Biochemical analysis of mutant RNA polymerase enzymes, including open complex formation at the lambda cro promoter.
- Assessment of the impact of mutations on transcription-coupled DNA repair and reactivation by GreA/GreB.
Main Results:
- 35 mutations identified, primarily altering single amino acids in RpoB or RpoC, located near the DNA path within RNA polymerase.
- Four mutant enzymes demonstrated unstable open complex formation at the lambda cro promoter.
- At least one mutant enzyme reduced the requirement for GreA/GreB and Mfd, suggesting altered stalled complex handling.
Conclusions:
- RNA polymerase mutations can mitigate defects caused by the absence of key DNA repair proteins.
- Mutations affecting RNA polymerase structure, particularly RpoB and RpoC, can influence transcription complex stability and DNA repair interactions.
- Findings illuminate the complex interplay between DNA replication, transcription, and DNA repair, offering insights into cellular conflict resolution.