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DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
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Break-induced replication orchestrates resection-dependent template switching.

Tianpeng Zhang1, Yashpal Rawal2, Haoyang Jiang1

  • 1Department of Cancer Biology, Penn Center for Genome Integrity, Basser Center for BRCA, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Nature
|June 14, 2023
PubMed
Summary
This summary is machine-generated.

Break-induced telomere synthesis (BITS) uses a minimal replisome for DNA repair. SNM1A nuclease directs ubiquitinated PCNA to promote resection and lesion bypass during this process.

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Area of Science:

  • Molecular Biology
  • DNA Repair
  • Telomere Biology

Background:

  • Break-induced telomere synthesis (BITS) is a RAD51-independent pathway crucial for alternative lengthening of telomeres.
  • BITS involves a minimal replisome, including proliferating cell nuclear antigen (PCNA) and DNA polymerase-δ, for extensive DNA repair synthesis.
  • The response of BITS to replication stress and secondary DNA structures is not well understood.

Purpose of the Study:

  • To investigate the DNA damage response during BITS under replication stress.
  • To identify key proteins involved in maintaining processivity during long-tract homologous recombination repair.
  • To elucidate the mechanism by which BITS tolerates complex DNA structures.

Main Methods:

  • Synchronous induction of double-strand breaks.
  • Proteomics of isolated chromatin segments (PICh) to analyze the telomeric DNA damage response proteome.
  • Analysis of RAD18-dependent PCNA ubiquitination and SNM1A nuclease activity.

Main Results:

  • BITS elicits a replication stress response characterized by RAD18-dependent PCNA ubiquitination.
  • The SNM1A nuclease is identified as a key effector of ubiquitinated PCNA-dependent DNA damage tolerance.
  • SNM1A recognizes ubiquitinated PCNA at the break-induced replisome, directing its nuclease activity to promote resection.

Conclusions:

  • Break-induced replication orchestrates resection-dependent lesion bypass.
  • SNM1A nuclease activity is critical for ubiquitinated PCNA-directed recombination in mammalian cells.
  • This study reveals a novel mechanism for DNA damage tolerance during BITS.