VirD2 of Agrobacterium tumefaciens : functional domains and biotechnological applications

Agrobacterium tumefaciens is a pathogenic bacterium, which can genetically transform plants and other organisms. It does so by translocating a part of its DNA, the T-strand, in complex with the relaxase protein VirD2. We have shown that VirD2 is the determinant of translocation of the VirD2-T-strand complex, rather than the T-strand. Furthermore, we have elucidated the role of the DUF domain of VirD2. DUF is shown to be important for localization of VirD2 to the cell poles. Polar localization is essential for translocation of VirD2 to the recipient cell. Using VirD2 as a carrier, we have shown that several biotechnological important enzymes can be transferred to Arabidopsis thaliana. Of the relaxase-homing endonuclease fusion protein VirD2-I-SceI, we have shown that it can induce the formation of double-strand breaks in the Arabidopsis genome after translocation from Agrobacterium.LEI Universiteit LeidenMoleculaire ontwikkelingsgenetic

Agrobacterium-mediated T-DNA transfer and integration by minimal VirD2 consisting of the relaxase domain and a type IV secretion system translocation signal.

Plant sciencesMicrobial Biotechnolog

Gene targeting in polymerase theta-deficient arabidopsis thaliana

Plant science

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

The repair of G-quadruplex-induced DNA damage

Toxicogenetic

T-DNA integration in plants results from polymerase-theta-mediated DNA repair

Genome Instability and Cance