Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair

Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase θ (Pol θ), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol θ contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol θ. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol θ can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol θ and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance

Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair

Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase theta (Pol theta), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol theta contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol theta. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol theta can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol theta and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance

Modeling biochemical pathways in the gene ontology.

The concept of a biological pathway, an ordered sequence of molecular transformations, is used to collect and represent molecular knowledge for a broad span of organismal biology. Representations of biomedical pathways typically are rich but idiosyncratic presentations of organized knowledge about individual pathways. Meanwhile, biomedical ontologies and associated annotation files are powerful tools that organize molecular information in a logically rigorous form to support computational analysis. The Gene Ontology (GO), representing Molecular Functions, Biological Processes and Cellular Components, incorporates many aspects of biological pathways within its ontological representations. Here we present a methodology for extending and refining the classes in the GO for more comprehensive, consistent and integrated representation of pathways, leveraging knowledge embedded in current pathway representations such as those in the Reactome Knowledgebase and MetaCyc. With carbohydrate metabolic pathways as a use case, we discuss how our representation supports the integration of variant pathway classes into a unified ontological structure that can be used for data comparison and analysis. Database (Oxford) 2016 Sep 1; 2016:baw126

Modeling biochemical pathways in the gene ontology

The concept of a biological pathway, an ordered sequence of molecular transformations, is used to collect and represent molecular knowledge for a broad span of organismal biology. Representations of biomedical pathways typically are rich but idiosyncratic presentations of organized knowledge about individual pathways. Meanwhile, biomedical ontologies and associated annotation files are powerful tools that organize molecular information in a logically rigorous form to support computational analysis. The Gene Ontology (GO), representing Molecular Functions, Biological Processes and Cellular Components, incorporates many aspects of biological pathways within its ontological representations. Here we present a methodology for extending and refining the classes in the GO for more comprehensive, consistent and integrated representation of pathways, leveraging knowledge embedded in current pathway representations such as those in the Reactome Knowledgebase and MetaCyc. With carbohydrate metabolic pathways as a use case, we discuss how our representation supports the integration of variant pathway classes into a unified ontological structure that can be used for data comparison and analysis

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

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

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