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

Macrophage differentiation: A heterogeneous theory

Functional heterogeneity of macrophage populations has been characterized however, questions remain regarding the mechanisms underlying the development of this heterogeneity. A procedure was developed to molecularly phenotype colonies of bone marrow-derived macrophage(s) (BMDM). Gene expression from individual clones was monitored using the reverse transcription polymerase chain reaction (RT-PCR) to permit simultaneous amplification of multiple specific gene transcripts. Internal "nested" primers were utilized in addition to the two traditional external primers thus, increasing the reliability of amplification. Results revealed hierarchal expression of macrophage-associated genes. Predominant colony phenotypes observed were unique both for the period of differentiation and choice of hematopoietic stimulus. Furthermore, colonies were subcloned into different growth factors and phenotyped on sequential days demonstrating that individual clones were not committed to specific phenotypes but retained the capacity to express different functional phenotypes.Tumor necrosis factor-$\alpha$ (TNF$\alpha$) transcripts were present in all colonies suggesting a role for this molecule during macrophage differentiation. Antisense oligomers to the initiation region of TNF$\alpha$ translation were utilized to inhibit TNF$\alpha$ expression and thus, determine its role in BMDM differentiation. GM-CSF-derived cells isolated on day 3 were exclusively vulnerable to inhibition of TNF$\alpha$ expression, displaying a 30% increase in proliferation over control values. Recombinant TNF$\alpha$ was able to rescue antisense-treated cells preventing increased proliferation. Furthermore, exogenous murine TNF$\alpha$ (mTNF$\alpha$) inhibited proliferation and differentiation of CSF-1-derived BMDM 50% while both human (hTNF$\alpha$) and mTNF$\alpha$ induced differentiation of GM-CSF-derived BMDM by 42%.Two distinct receptors for TNF (TNFR; designated p60 and p80) are distinguished by their species specificity for TNF$\alpha$ binding. TNFR p60 binds both hTNF$\alpha$ and mTNF$\alpha$ whereas p80 exclusively binds mTNF$\alpha$. Northern blot analysis demonstrated similar expression of p60 and p80 in CSF1- and GM-CSF-derived BMDM. Results suggest differential signalling in GM-CSF- and CSF-1-derived macrophages with p80 functioning in TNF$\alpha$-mediated growth inhibition of CSF-1-derived progenitors and p60 functioning in TNF$\alpha$-induced differentiation of GM-CSF-derived BMDM.U of I OnlyETDs are only available to UIUC Users without author permissio

Removal of the Bloom Syndrome DNA Helicase Extends the Utility of Imprecise Transposon Excision for Making Null Mutations in Drosophila

Transposable elements are frequently used in Drosophila melanogaster for imprecise excision screens to delete genes of interest. However, these screens are highly variable in the number and size of deletions that are recovered. Here, we show that conducting excision screens in mus309 mutant flies that lack DmBlm, the Drosophila ortholog of the Bloom syndrome protein, increases the percentage and overall size of flanking deletions recovered after excision of either P or Minos elements

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 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