CHARACTERIZATION OF ATG8 GENE HOMOLOGS IN VERTICILLIUM DAHLIAE AND VERTICILLIUM ALBO-ATRUM

The vascular wilt fungi Verticillium dahliae and the closely related Verticillium albo-atrum are devastating plant pathogens. Both pathogens produce resting structures that accumulate in soil, and are difficult to eradicate. V dahliae produces microsclerotia (MCS), while V albo-atrum produces dark resting mycelia (DRM). The role ofATG8, an autophagy marker was studied by generating A TG8 knockouts in V dahliae (vdatg8), and V albo-atrum (vaatgS). Although dispensable for pathogenicity in both species, in V dahliae ATG8 was involved in dimorphic growth, conidiation, and MCS formation, but not glycogen accumulation. Increased temperatures restored conidiation and MCS formation in vdatg8, indicating that autophagy is involved in, but not essential for MCS formation in V dahliae. In V albo-atrum ATG8 was involved in glycogen accumulation, but not DRM formation. Considerable functional redundancy exists in V dahliae MCS formation, and although VdATG8 and VaATG8 amino acid sequences are almost identical, A TG8 function is species specific

FANCL (FA complementation group L)

Review on FANCL, with data on DNA, on the protein encoded, and where the gene is implicated

Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin.

Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways