Due to the action of endogenous and
exogenous agents, DNA is subject up to 70,000 lesions per day, thus the
existence of repair mechanisms and enzymes is more than necessary. We already
know basic mechanisms of several specific DNA repair pathways include the base
excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR),
homologous recombination (HR), and among others, Fanconi anaemia (FA) repair
pathway[1]. FA is a rare, autosomal-recessive
disorder characterized by early onset bone marrow failure, developmental
defects, genomic instability and predisposition to acute myeloid leukaemia and
solid tumours. The primary diagnosis of FA is a hypersensitivity to cross-linking
agents such as mitomycin C or cisplatin. The molecular defect in FA is an
impaired repair of DNA interstrand cross-links (ICLs). The ICLs are cytotoxic
lesions that inhibit process of DNA replication and transcription. Crucial step
in FA pathway that initiates ICL repair is a mono-ubiquitination of FANCD2 and
FANCI. Ubiquitinated FANCD2-FANCI is a base for the recruitment of additional
proteins that coordinate DNA repair. Ubiquitin (Ub) is recruited via activating
enzyme E1 (UBA1), ubiquitin conjugating enzyme 2T (UBE2T) and transferred onto
FANCD2 resp. FANCI by multisubunit E3 ligase (FA core complex). There is upto 11
different proteins composing FA core complex whilst most important are FANCB,
FAAP100 and FANCL[2,3]. However, minimal ubiquitination
system for proper
mono-ubiquitination of FANCD2 and FANCI is ubiquitin, UBE2T, FANCL, UBA1 and
DNA. In this work we study molecular architecture and mechanistic properties
regulating the ubiquitination and entire FA pathway. We have produced covalent complex
of UBE2T with ubiquitin bound by thioester bond.
In order to investigate the molecular mechanisms of mono-ubiquitination of
FANCD2 we have prepared variants of constructs of all three different domains
of FANCL. These were expressed, purified and we have assayed their enzymatic
activities and binding properties. We obtained minimal components for
sufficient FANCD2 mono-ubiquitination. These were put forward in experiments
focused on crystallization of complex UBE2T-Ub with FANCL. Our aim is to obtain
structural information
about interaction sites of ubiquitin in complex with UBE2T and FANCL for better
understanding of molecular mechanism of activation step in FA repair pathway.
Since ICL-forming agents are used in cancer therapy, our structure could help
in development of therapeutic targets for inhibiting repair mechanism in cancer
cells.
The project was supported by the Czech Science Foundation (17-21649Y). We thank to Academy of Sciences Czech Republic for support in form of J.E. Purkyne Fellowship awarded to J.S. The project was also supported by the Academy of Sciences Czech Republic (RVO: 61388963).