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Related Concept Videos

Homologous Recombination02:31

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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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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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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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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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Crossing over is the exchange of genetic information between homologous chromosomes during prophase I of meiosis I. Genetic recombination gives rise to allelic diversity in the newly formed daughter cells. In humans, crossing over produces genetically distinct haploid egg and sperm cells that undergo fertilization to produce unique offspring. Before cell division starts, the germ cell’s chromosome(s) undergo duplication in the S phase of the cell cycle. As the cells enter prophase I,...
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In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
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TLK1-mediated RAD54 phosphorylation spatio-temporally regulates Homologous Recombination Repair.

Ishita Ghosh1, Youngho Kwon2, Aida Badamchi Shabestari2

  • 1Department of Biochemistry and Molecular Biology, Louisiana Health Science Center-Shreveport, Shreveport, Louisiana 71130, US2. Texas 78229, USA.

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Tousled-like kinase 1 (TLK1) regulates DNA repair by phosphorylating RAD54. Phosphorylation at T41 and T59 aids homologous recombination repair (HRR), while T700 phosphorylation impairs HRR and delays repair.

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Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Genetics

Background:

  • Environmental agents induce DNA double-strand breaks (DSBs), critical lesions that can cause cell death.
  • Homologous recombination repair (HRR) is a major error-free pathway for repairing DSBs.
  • Tousled-like kinase 1 (TLK1) is a key regulator of the DNA damage checkpoint and interacts with RAD54, a crucial protein in HRR.

Purpose of the Study:

  • To investigate the regulatory mechanism of RAD54 by TLK1 in human cells.
  • To determine the specific sites of RAD54 phosphorylation by TLK1 and their functional consequences on HRR.

Main Methods:

  • TLK1 was inhibited or depleted in human cells.
  • HRR activity was assessed using a reporter system (ISce-I-GR-DsRed).
  • RAD54 phosphorylation sites were identified and analyzed.

Main Results:

  • TLK1 phosphorylates RAD54 at three threonine residues: T41, T59 (N-terminal domain), and T700 (C-terminal domain).
  • Phosphorylation of T41 and T59 enhances HRR and confers protection against DSB-induced cytotoxicity.
  • Phosphorylation of T700 impairs HRR, delays repair, and offers no protection from cytotoxicity.

Conclusions:

  • TLK1-mediated phosphorylation of RAD54 at T41 and T59 is essential for efficient HRR and cellular survival following DSB damage.
  • TLK1-mediated phosphorylation of RAD54 at T700 has detrimental effects on HRR, highlighting a complex regulatory role.
  • This study reveals a novel interaction site between RAD51 and the TLK1-phosphorylated RAD54 C-terminal domain.