Structural basis for a novel mechanism of DNA bridging and alignment in eukaryotic DSB DNA repair

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• Eukaryotic DNA polymerase mu (Pol mu) facilitates DNA double-strand break (DSB) repair via non-homologous end-joining (NHEJ) by promoting DNA synapsis.
• Terminal deoxynucleotidyltransferase (TdT) is a related PolX enzyme crucial for V(D)J recombination.

Purpose of the Study:

• To investigate the DNA synapsis capabilities of TdT, a PolX family member.
• To elucidate the structural mechanisms by which TdT bridges DNA ends during recombination.
• To determine if the structural model for TdT applies to Pol mu in NHEJ.

Main Methods:

• X-ray crystallography to solve the structure of TdT bound to a DNA synapsis.
• Site-directed mutagenesis to probe protein-DNA interactions.
• Functional assays to assess DNA repair and recombination activities.

Main Results:

• TdT, similar to Pol mu, can promote DNA synapsis independently of core NHEJ machinery.
• The crystal structure reveals TdT's N-terminal domain and Loop 1 cooperate to bridge DNA ends.
• TdT provides a templating base in trans and limits micro-homology search to two base pairs.
• A network of water molecules may facilitate nucleotide incorporation independent of the templating base.
• The structural model is consistent with in vivo TdT sequence synthesis and applicable to Pol mu in NHEJ.

Conclusions:

• TdT and Pol mu share the ability to facilitate DNA end bridging, a key step in DSB repair and V(D)J recombination.
• The solved TdT structure provides a mechanistic basis for its role in templating and micro-homology recognition.
• The findings suggest a conserved structural mechanism for DNA end association across different PolX enzymes and repair pathways.