Abstract
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.
Highlights
DNA polymerases (Pols) are essential for life since they are necessary for the propagation and maintenance of genetic information
A current paradox in our understanding of alternative end-joining (alt-EJ) is that polymerase q (Polq) promotes non-templated nucleotide insertions at DNA repair junctions in vivo, but lacks template-independent terminal transferase activity in vitro
This shows that efficient single-strand DNA (ssDNA) extension by Polq requires the complementary nucleotide, which demonstrates that the template bases facilitate the nucleotidyl transferase reactions by pairing with the incoming nucleotide
Summary
DNA polymerases (Pols) are essential for life since they are necessary for the propagation and maintenance of genetic information. Bacterial and eukaryotic cells encode for multiple different types of Pols, some of which are intrinsically error-prone due to their relatively open active sites which enables them to tolerate particular DNA lesions (Foti and Walker, 2010; Lange et al, 2011; Sale et al, 2012; Waters et al, 2009) Such enzymes are referred to as translesion polymerases and are mostly among the Y-family of polymerases (Foti and Walker, 2010; Lange et al, 2011; Sale et al, 2012; Waters et al, 2009). In recent studies we demonstrated the ability of the polymerase domain of POLQ, referred to as Polq, to perform microhomology-mediated end-
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
