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

Distortion in formalin-fixed brains: Using geometric morphometrics to quantify the worst-case scenario in mice

Although morphometric studies of fixed mammalian brains are an integral part of neuroscience, the nature of fixation-related morphometric artifacts is not well understood beyond assessments of size changes over fixation time. This study is the first to quantitatively co-evaluate the effects of the most common brain tissue fixative-formalin-on brain shape, size, and weight, using two-dimensional landmark analysis of mouse brains fixed in unbuffered, non-saline formalin from fresh specimens up to 213 days of preservation. The brains show a typical swelling reaction with subsequent decline in size and weight. Weight initially under- and later over-estimates size, so that the practice of using weight to estimate volume can be problematic. Time to recovery of original size resembled that of much larger brained mammals, suggesting that the slow reaction of formalin with tissue components mainly determines recovery times. Non-size related (anisotropic) distortion of different brain areas accounted for around a quarter of overall change suggesting that the use of "all-brain" fixation correction factors can introduce considerable error. Distortion occurs mostly after the first day of fixation, and extended fixation times impact mostly on size, not shape. Fixation effects relatively wider and stouter brain dimensions, except the cerebellum whose shape changes less. Evidence from the literature suggests that this pattern may be common to mammals due to structural commonalities