ATPase-dead <i>P{w</i>^<i>a</i><i>}</i> repair junctions.

<p>ATPase-dead <i>P{w</i>^<i>a</i><i>}</i> repair junctions.</p

Efficient bypass of interstrand crosslinks by Pol θ.

<p><b>A</b>. Structures of a nitrogen mustard interstrand crosslink (ICL) between two guanines and a 5-atom synthetic nitrogen mustard ICL mimic. <b>B</b>. Substrates used in the primer extension assays. 6-FAM labeled primer was annealed to different templates: (i.) single-stranded control containing ICL precursor G, (ii.) double-stranded control with ICL precursor G, (iii.) ICL substrate in a 6 bp duplex, (iv.) ICL substrate in a 20 bp duplex. Red highlighting indicates ICL precursor G (G_OH) or crosslinks. <b>C</b>. Comparison of ICL bypass between Klenow fragment (KF), wild-type Pol θ (WT), and ATPase-dead Pol θ (AD). 5 nM of control or ICL templates were incubated with 1 nM Klenow for 5 min or 0.2 nM Pol θ for 10 min. at 37°C. Products were separated by denaturing PAGE on a 10% gel. <b>D</b>. Quantification of bypass. Each lane was divided into approach (-14 to -1), insertion (0) and bypass (+1 to +8) segments and corresponding band intensities expressed as a percentage of all the products combined. Data represent the mean of three experiments and error bars indicate standard deviations.</p

Pol θ ATPase activity does not affect end joining frequency but promotes the formation of complex insertions.

<p><b>A</b>. Schematic of the <i>P{w</i>^<i>a</i><i>}</i> construct (top). After expression of transposase, the P-element is excised leaving 17 nt overhangs (bottom). <b>B</b>. Frequency of end joining repair in domain-specific transgenic alleles and controls. All transgenes are in a <i>mus308Δ</i>, <i>spn-A</i> (<i>rad51</i>) background. The number of independent vials (n) represented by each bar and the standard error of the mean is shown. <b>C</b>. Summary of junction types recovered in control and ATPase-dead transgenic alleles. *p<0.05, **p<0.01, # p = n.s. compared to wild-type control, two-way ANOVA, Tukey’s post hoc test.</p

Pol θ ATPase activity is important for annealing and extension reactions <i>in vitro</i>.

<p><b>A.</b> Pol θ promotes annealing of partial single-stranded DNA (pssDNA) at terminal microhomologies and DNA synthesis. 26 nt pssDNA with a CCGG terminal microhomology is from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006813#pgen.1006813.ref012" target="_blank">12</a>]. 30 nM of pssDNA was incubated with 50 pM of Klenow fragment (KF) or wild-type (WT), ATPase-dead (AD), or Pol-dead (PD) Pol θ protein for 30 min at 37°C. <b>B.</b> Pol θ can promote inter- and intra-molecular annealing and extension reactions on pss and ssDNA. 33 nt pssDNA with a TA terminal microhomology corresponds to the DNA product created by <i>P{w</i>^<i>a</i><i>}</i> excision. 33 nt ssDNA is the top strand of 33 nt pssDNA. 30 nM of pssDNA or ssDNA was incubated with 50 pM of protein for 30 min at 37°C. All products were separated by denaturing PAGE on a 20% gel. Percent extension was calculated by measuring band intensities of all primer extension products and dividing by total intensity of all bands in the lane.</p