Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining.
DNA polymerase θ (Polθ) promotes insertion mutations during alternative end-joining (alt-EJ) by an unknown mechanism. Here, we discover that mammalian Polθ transfers nucleotides to the 3' terminus of DNA during alt-EJ in vitro and in vivo by oscillating between three different modes of terminal transferase activity: non-templated extension, templated extension in cis, and templated extension in trans. This switching mechanism requires manganese as a co-factor for Polθ template-independent activity and allows for random combinations of templated and non-templated nucleotide insertions. We further find that Polθ terminal transferase activity is most efficient on DNA containing 3' overhangs, is facilitated by an insertion loop and conserved residues that hold the 3' primer terminus, and is surprisingly more proficient than terminal deoxynucleotidyl transferase. In summary, this report identifies an unprecedented switching mechanism used by Polθ to generate genetic diversity during alt-EJ and characterizes Polθ as among the most proficient terminal transferases known.
Fels Institute for Cancer Research, Temple University Lewis Katz School of Medicine, Philadelphia, United States.|Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, United States.
Tatiana
T
Kent
Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, United States.|Department of Cell Biology, New York University School of Medicine, New York, United States.
Pedro A
PA
Mateos-Gomez
Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, United States.|Department of Cell Biology, New York University School of Medicine, New York, United States.
Agnel
A
Sfeir
Fels Institute for Cancer Research, Temple University Lewis Katz School of Medicine, Philadelphia, United States.|Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, United States.
Richard T
0000-0003-1194-9871
RT
Pomerantz
D000818:Animals; D003067:Coenzymes / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D059766:DNA End-Joining Repair; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D053595:Embryonic Stem Cells; D008345:Manganese / Q000378:metabolism; D051379:Mice; D000097462:DNA Polymerase theta
D003067:Coenzymes; D004247:DNA; D004253:DNA Nucleotidylexotransferase; D004259:DNA-Directed DNA Polymerase; D008345:Manganese; D000097462:DNA Polymerase theta
DNA polymerase; DNA repair; alternative end-Joining; biochemistry; chromosomes; genes; genetic diversity; genetic instability; human; microhomology-mediated end-joining; mouse