Model for polymerase action at a DSB.

<p>Multiple polymerases compete for access to D-loops. Following formation of a double-strand gap, Rev1 binds at the break site(s), recruits pol zeta, and blocks access of other polymerases. Initial synthesis is carried out by pol zeta, which readily dissociates. Repair can then be completed by end joining or another polymerase can bind and reinitiate synthesis. Binding of pol delta and its processivity subunit Pol32 to the D-loop results in processive synthesis and promotes repair of large gaps. Other polymerases, including pol eta, can act in backup roles. Elimination of Rev1 or multiple TLS polymerases increases the probability of pol delta recruitment leading to increased repair synthesis.</p

<i>pol32</i> mutants are sensitive to multiple DNA damaging agents.

<p>(A) A null allele (<i>L2</i>) of <i>POL32</i> (<i>CG3975</i>) was created through imprecise excision of a <i>P</i> element (EY15283) located in the 3′ untranslated region (UTR) of the <i>POL32</i> gene. White box indicates the <i>POL32</i> open reading frame; shaded regions, the UTRs; numbers indicate nucleotide position from start of transcription. (B) <i>pol32</i> mutants are sensitive to ionizing radiation (IR). Percent survival was calculated as the percentage of homozygote eclosion relative to an untreated control. (C) <i>pol32</i> mutants are sensitive to methyl methanesulfanate (MMS) and nitrogen mustard (HN2), but not camptothecin (CPT). Error bars represent the standard deviation for at least three trials.</p

<i>pol32</i> mutants are impaired in DNA synthesis during HR repair.

<p>(A) The <i>P{w^a}</i> site-specific repair assay. Expression of transposase in males possessing <i>P{w^a}</i> (i) results in a 14 kilobase gap (ii) relative to an uncut sister chromatid. Full HR requires synthesis of the <i>white</i> gene and <i>copia</i> long terminal repeats (LTRs), followed by annealing at the LTRs (iii). Aborted HR results when end-joining repair occurs prior to synthesis of the entire <i>white</i> gene. Amount of repair synthesis in aborted HR repair events can be estimated by PCR (iv). (B) <i>pol32</i> mutants are significantly impaired in full HR repair relative to wildtype. Wildtype n = 55; <i>pol32</i> n = 120. Error bars represent standard errors. *<i>P</i><0.05, Mann-Whitney test. (C) Repair synthesis is decreased in <i>pol32</i> mutants. Each bar represents the percentage of events with at least the indicated amount of synthesis. Right end: wildtype n = 55; <i>pol32</i> n = 151. Left end: wildtype n = 55 <i>pol32</i> n = 66. *<i>P</i><0.05, Fisher's exact test.</p

Rev1 regulates extent of repair synthesis during HR.

<p>(A) Left panel: HR efficiency is unchanged in <i>rev1</i> mutants (n = 104). Right panel: repair synthesis is increased in <i>rev1</i> mutants (n = 97). *<i>P</i><0.05, Fisher's exact test. (B) Left panel: flies lacking both Pol32 and Rev3 have decreased full HR repair (n = 173). **<i>P</i><0.01, Mann Whitney test. Right panel: aborted HR events have shorter synthesis tract lengths in <i>pol32 rev3</i> mutants (n = 45). (C) Left panel: flies lacking both Pol32 and Rev1 have decreased full HR repair (n = 34). *<i>P</i><0.05, Mann-Whitney test. Right panel: aborted HR events from <i>pol32 rev1</i> mutants (n = 45) have increased synthesis tract lengths compared to wildtype.</p

Flies lacking pol eta and the catalytic subunit of pol zeta are HR–deficient.

<p>(A) Flies lacking Rev3, but not pol eta, are sensitive to nitrogen mustard (HN2) and ionizing radiation (IR). (B) Both <i>pol eta</i> and <i>rev3</i> mutants have decreased full HR repair. Wildtype n = 43; <i>pol eta</i> n = 38; <i>rev3</i> n = 98. *<i>P</i>≤0.05, Mann-Whitney test. Wildtype versus <i>rev3</i> aborted HR repair, <i>P</i> = 0.062, Mann-Whitney test. (C) Repair synthesis is unchanged in the absence of pol eta (n = 19) or Rev3 (n = 38). (D) Left panel: <i>pol eta rev3</i> double mutants have no change in full HR, but a decrease in aborted HR (n = 85). *<i>P</i><0.05, Mann-Whitney test. Right panel: repair synthesis tract lengths are increased in <i>pol eta rev3</i> mutants (n = 24). *<i>P</i><0.05, Fisher's exact test.</p

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

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

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