θPD.(A) AlphaFold rendering of zPol θ PD with subdomains colored, thumb residues 2093-2217 – blue, fingers residues 2327-2468 – red, palm – residues 2217-2252; 2469-2576 green, and exo-nuclease residues 1832-2093 – yellow. DNA is colored in light blue. (B) FATCAT overlay of AlphaFold rending of zPol θ PD (bronze) and hPol θ PD (navy) crystal structure [21] without loop inserts. DNA is colored in light blue. PDB 4X0Q.

(A) SDS-PAGE of purified hPol θ PD and zPol θ PD. Expression and purification of zPol θ PD was the same as hPol θ PD as described in the Materials and Methods. For each sample, approximately 56–60 pmol of cleaved, purified protein were loaded on a 10% SDS PAGE and Coomassie stained. Both hPol θ and zPol θ PD fragments migrate to approximately 90 kDa as expected. (B) Circular dichroism spectra of 3 μM hPol θ PD (solid line) and zPol θ PD (dashed) proteins in 10 mM Potassium Phosphate buffer. Samples were scanned from 190 to 280 nm. (C) The same samples were heated from 20–90°C and ellipticity measured at 222 nm.

zPol θ PD was titrated from 0–1000 nM against 10 nM 25/40 dsDNA. Bound and unbound products were separated on a 6% non-denaturing gel and quantified using ImageQuant. K_D(DNA) was mathematically calculated using Equation 1 and is the midpoint between bound and unbound fractions.

Denaturing gel showing primer extension dsDNA substrate with MgCl₂ and MnCl_2. (A) Under steady-state conditions 50 nM hPol θ or zPol θ PD proteins were preincubated with 200 nM 25/40 dsDNA and combined with either 10 mM MgCl₂ or MnCl₂ for 5 minutes at 37°C. (B) The assay was repeated under steady-state conditions, with 10 nM Pol θ and 25 nM 25/40 dsDNA. Reactions were initiated with 100 µM dNTPs, 10 mM MgCl₂ or MnCl₂, and 75-fold excess of unlabeled 25/40 dsDNA for 5 minutes at 37°C (indicated by a *). DNA extension products were separated on a denaturing gel and visualized on a Typhoon scanner. Each n+1 band represents an extension of one nucleotide following the DNA template as described above. n+1 would represent either correct nucleotide incorporation of dCTP opposite a templating G (underlined) or a misincorporation event of dATP, dGTP, or dTTP opposite templating G. Each subsequent band is another nucleotide extension with a maximum template-dependent extension of n+18. Bands migrating higher than n+18 represent de novo synthesis.

ZPol θ PD (100 nM) was preincubated with 300 nM 25/40 dsDNA. The DNA/Pol θ PD complex was rapidly combined with 100 μM dCTP (correct nucleotide) and 10 mM MgCl₂. Reactions were carried out at 37°C and quenched with 0.5 M EDTA. Products were separated on a denaturing gel and quantified with ImageQuant software. Data were fit to a biphasic burst equation to obtain observed k_obs rates 15.9 ± s^-1. The slower rate k_ss was calculated to be 3.4 ± 0.46 s^-1.

Non-Denaturing gel demonstrating alignment and extension of ssDNA. zPol θ PD (20 nM) was preincubated with 30 nM 5’-FAM 12-mer ssDNA in reaction buffer. Samples either had all nucleotides (+dNTP) or no nucleotides (-dNTP) added and the ternary complex was incubated for 45 minutes at 37°C. Reactions were stopped and products separated on a 12% Native PAGE. The gel was visualized on a Typhoon scanner. MMEJ products are indicated, as well as smaller snap-back products indicated by the bracket.

Denaturing gel demonstrating primer extension on damaged dsDNA substrates with MgCl₂ and MnCl_2.As described in the Materials and Methods, 200 nM hPol θ and zPol θ PDs were preincubated with 50 nM DNA substrate, (A) Undamaged (TT), (B) CPD (TT), (C) Abasic ((AP)T), (D) 8-oxo-dG ((8-oxo-dGT)T). Reactions were initiated by the addition of 125 nM dNTPs as described and either 10 mM MgCl₂ or MnCl₂. Reactions were carried out at 37°C for 5 minutes and products visualized on a 12% denaturing gel. Higher migrating products are indicative of full extension (n+12) with smeared bands representing de novo synthesis with extension past n+12.

θPD is able incorporate and extend dsDNA in the presence of Ca²⁺. Denaturing gel showing primer extension dsDNA substrate with CaCl_2. Under single turnover conditions 200 nM Pol θ PD (human or zebrafish) was preincubated with 50 nM 24/33 undamaged (A) or damaged (B) DNA substrate and reacted with 125 nM nucleotides in 10 mM CaCl₂. Reactions were carried out at 37°C for 5 minutes and products visualized on a 12% denaturing gel. Percent extended was calculated using ImageQuant software by quantifying the intensity of the extended products (n+1 and higher) divided by the intensity of the total amount of DNA.