Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining

• Published in: eLife Vol. 5
• Main Authors: Kent, Tatiana, Mateos-Gomez, Pedro A, Sfeir, Agnel, Pomerantz, Richard T
• Format: Journal Article
• Language: English
• Published: England eLife Science Publications, Ltd 17-06-2016; eLife Sciences Publications Ltd; eLife Sciences Publications, Ltd
• Subjects: alternative end-Joining; Animals; Biochemistry; Cloning; Coenzymes - metabolism; Deoxyribonucleic acid; DNA; DNA - metabolism; DNA End-Joining Repair; DNA nucleotidylexotransferase; DNA Nucleotidylexotransferase - metabolism; DNA polymerase; DNA Polymerase theta; DNA polymerases; DNA repair; DNA sequencing; DNA-directed DNA polymerase; DNA-Directed DNA Polymerase - metabolism; E coli; Embryonic Stem Cells; Enzymes; Gene mutations; Genes and Chromosomes; Genetic diversity; genetic instability; Insertion; Kinases; Manganese; Manganese - metabolism; Medicine; Methods; Mice; microhomology-mediated end-joining; Mutagenesis; Nucleotide sequencing; Nucleotides; Observations; Physiological aspects; genetic instability; genetic diversity; mouse; DNA polymerase; microhomology-mediated end-joining; genes; biochemistry; chromosomes; DNA repair; alternative end-Joining; human
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.13740

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.