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An additional replication origin causes cell cycle specific DNA replication fork speed.

Ole Skovgaard1

  • 1Department of Science and Environment, Roskilde University, Roskilde, Denmark.

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Replication fork speed (RFS) in Escherichia coli varies dynamically, not constant as previously thought. Nucleotide supply limits RFS, impacting DNA replication kinetics.

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DNA replicationDnaAcell cycledNTP poolmarker frequency analysisoriCreplication fork speed

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Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • Replication fork speed (RFS) in Escherichia coli was traditionally considered constant.
  • Bidirectional replication from oriC normally results in two replication forks.
  • Cellular resources are strained under slow-growth conditions, impacting DNA replication.

Purpose of the Study:

  • To investigate the dynamic nature of RFS within the cell cycle.
  • To challenge the paradigm of constant RFS in Escherichia coli.
  • To explore the impact of altered replication fork numbers on RFS.

Main Methods:

  • Utilized an Escherichia coli strain with an additional ectopic oriC (oriX).
  • Analyzed DNA replication initiation from both oriC and oriX, creating up to four forks.
  • Calculated RFS using marker frequency analysis of deep sequencing data.

Main Results:

  • RFS decreased by approximately one third with four active forks and increased by one fourth with one active fork.
  • A 2-fold variation in RFS was observed during the replication cycle.
  • Delaying initiation or increasing dNTP pool normalized RFS variations.

Conclusions:

  • RFS is not constant within a replication cycle and varies dynamically.
  • Nucleotide supply is a primary constraint on replication fork speed.
  • Findings provide a basis for studying factors regulating DNA replication kinetics.