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Two components of DNA replication-dependent LexA cleavage.

Kamila K Myka1, Kenneth J Marians2

  • 1Molecular Biology Program, Sloan Kettering Institute Memorial Sloan Kettering Cancer Center, New York, New York USA.

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|June 10, 2020
PubMed
Summary

Bacterial DNA damage response (SOS) relies on DNA replication generating single-stranded DNA (ssDNA). RecA protein binding to ssDNA triggers LexA repressor cleavage, activating DNA repair genes.

Keywords:
DNA damage responseDNA replicationE. coliEscherichia coli (E. coli)LexASOS responseenzyme degradationgene regulationproteolytic cleavagesingle-stranded DNA

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Area of Science:

  • Bacteriology
  • Molecular Biology
  • Genetics

Background:

  • The SOS response in bacteria is a crucial DNA damage repair mechanism.
  • This response is initiated by the formation of single-stranded DNA (ssDNA) during replication.
  • RecA protein binding to ssDNA is essential for activating downstream repair pathways by cleaving the LexA repressor.

Purpose of the Study:

  • To investigate the mechanisms of LexA cleavage during DNA replication of both undamaged and damaged DNA templates.
  • To identify the specific DNA structures and proteins involved in SOS induction during replication.

Main Methods:

  • Utilized an in vitro reconstituted DNA replication system.
  • Performed LexA cleavage assays using both undamaged and base-damaged DNA templates.
  • Analyzed the role of RecA, ssDNA, and RecF in LexA cleavage during replication.

Main Results:

  • LexA cleavage was dependent on the formation of ssDNA-RecA filaments.
  • Replication of undamaged DNA induced LexA cleavage comparable to damaged templates.
  • Two pathways generating ssDNA were identified: polymerase-helicase uncoupling and replisome lesion skipping, with RecF specifically aiding the latter.

Conclusions:

  • ssDNA generation during normal replication and due to replication stress both contribute to SOS induction.
  • RecF protein plays a specific role in facilitating RecA filament formation on ssDNA gaps arising from replisome lesion skipping on damaged DNA.