DNA polymerase θ specializes in incorporating synthetic expanded-size (xDNA) nucleotides.

DNA polymerase θ (Polθ) is a unique A-family polymerase that is essential for alternative end-joining (alt-EJ) of double-strand breaks (DSBs) and performs translesion synthesis. Because Polθ is highly expressed in cancer cells, confers resistance to ionizing radiation and chemotherapy agents, and promotes the survival of homologous recombination (HR) deficient cells, it represents a promising new cancer drug target. As a result, identifying substrates that are selective for this enzyme is a priority. Here, we demonstrate that Polθ efficiently and selectively incorporates into DNA large benzo-expanded nucleotide analogs (dxAMP, dxGMP, dxTMP, dxAMP) which exhibit canonical base-pairing and enhanced base stacking. In contrast, functionally related Y-family translesion polymerases exhibit a severely reduced ability to incorporate dxNMPs, and all other human polymerases tested from the X, B and A families fail to incorporate them under the same conditions as Polθ. We further find that Polθ is inhibited after multiple dxGMP incorporation events, and that Polθ efficiency for dxGMP incorporation approaches that of native dGMP. These data demonstrate a unique function for Polθ in incorporating synthetic large-sized nucleotides and suggest the future possibility of the use of dxG nucleoside or related prodrug analogs as selective inhibitors of Polθ activity.

Identification of a Small Interface between the Methyltransferase and RNA Polymerase of NS5 that is Essential for Zika Virus Replication.

The spread of Zika virus (ZIKV) has caused an international health emergency due to its ability to cause microcephaly in infants. Yet, our knowledge of how ZIKV replicates at the molecular level is limited. For example, how the non-structural protein 5 (NS5) performs replication, and in particular whether the N-terminal methytransferase (MTase) domain is essential for the function of the C-terminal RNA-dependent RNA polymerase (RdRp) remains unclear. In contrast to previous reports, we find that MTase is absolutely essential for all activities of RdRp in vitro. For instance, the MTase domain confers stability onto the RdRp elongation complex (EC) and and is required for de novo RNA synthesis and nucleotide incorporation by RdRp. Finally, structure function analyses identify key conserved residues at the MTase-RdRp interface that specifically activate RdRp elongation and are essential for ZIKV replication in Huh-7.5 cells. These data demonstrate the requirement for the MTase-RdRp interface in ZIKV replication and identify a specific site within this region as a potential site for therapeutic development.

Modification of 3' Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity.

DNA polymerase θ (Polθ) is a promiscuous enzyme that is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ). During this form of DSB repair, Polθ performs terminal transferase activity at the 3' termini of resected DSBs via templated and non-templated nucleotide addition cycles. Since human Polθ is able to modify the 3' terminal ends of both DNA and RNA with a wide array of large and diverse ribonucleotide and deoxyribonucleotide analogs, its terminal transferase activity is more useful for biotechnology applications than terminal deoxynucleotidyl transferase (TdT). Here, we present in detail simple methods by which purified human Polθ is utilized to modify the 3' terminal ends of RNA and DNA for various applications in biotechnology and biomedical research.

The helicase domain of Polθ counteracts RPA to promote alt-NHEJ.

Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Polθ-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.