The Structural And Functional Research Of DNA Damage Repair Related Complexes FANCM-MHF And Shu

The stability of genome is the prerequisite of every life form for existence and continuation. As the basic repository of the genetic material, DNA, however, does not guarantee such requirement due to its physicochemical constitution. Environmental agents such as the ultraviolet, ionizing radiation and a variety of drugs used in chemotherapy can induce alterations in DNA structure. Moreover, numerous damaged DNA intermediates could arise spontaneously during normal cellular metabolism such as DNA replication and recombination. Unless repaired promptly and correctly, the DNA lesions in the genes would lead to disastrous consequences-cell death and oncogenesis. To combat those threats, organism have evolved several mechanisms to resist all kinds of DNA lesions such as nucleotide excision repair and homologous recombination repair.During cell division, DNA interstrand crosslinks (ICLs) are most dangerous as such lesions would block the progression of replication fork completely. Failing to replicate DNA properly, cells thus could not to divide. Fanconi anemia (FA) pathway in the human cells, mediated by a complementary group of proteins, specifically deal with such DNA lesions. To date,15FA proteins have been identified, of which mutations in any would cause FA. In addition to these FA proteins, there are several FA-associated factors, which are essential for activation of the FA pathway. FANCM, as a component of FA core complex, forms a complex with two factors, MHF1and MHF2, and this complex constitutively localizes to chromatin. The FANCM/MHF1/MHF2complex specifically recognizes the ICLs and recruits FA core complex to assemble at chromatin.In this work, we targeted FACNM/MHF1/MHF2complex for structural studies and determined the crystal structures of the MHF1/MHF2complex alone and bound to FANCM661-800. The structures show that both MHF1and MHF2adopt a histone-fold, which mediates the MHF heterodimer in a head-to-tail fashion, and the MHF heterodimer further assemble into (MHF1-MHF2)2tetramer. In relation to (H3-H4)? tetramer, MHF tetramer has a highly compact structure induced by a serious of intra-molecular hydrophobic interactions and the unique tetramer interface. Immunoprecipitation and immunofluorescence reveal that (MHF1-MHF2)2tetramer is the functional unit, whose mutation would disrupt the assembly of FANCM/MHF1/MHF2complex. FANCM binds to the (MHF1-MHF2)2tetramer through a’dual-V shaped structure and constructs an extensive interface. FANCM and MHF bind to DNA synergistically and cooperate to constitute a new DNA-binding site that is coupled to the canonical L1L2region of (MHF1-MHF2)2tetramer. By association with DNA,(MHF1-MHF2)2tetramer localizes FANCM to centromeres. A disease-associated mutant of FANCM (FANCMS724X), only retaining the N-terminal "V" structure, could not interact with MHF and its subcellular localization is altered, which may partially explain the pathogenesis of such mutations in FA patients.In addition to the various precise DNA repair pathways, all cells have evolved a pathway, namely DNA-damage-tolerance (DDT), to complete DNA replication without the actual removal of the damage. DDT is a highly conserved mechanism and exists in both prokaryotes and eukaryotes. In Saccharomyces cerevisiae, DDT can be divided into two parallel pathways, one being error-prone (TLS) and another error-free. Monoubiquitination of the PCNA activates TLS and polyubiquitinated PCNA switches the damage response into error-free DDT. In the presence of homologous recombination machine, error-free DDT has to utilize newly synthesized sister chromatid as a template to bypass the lesions, but how it is achieved remains unknown. Shu complex from budding yeast was identified to function in error-free DDT and couple it to homologous recombination. Shu complex is a regulator of the anti-recombinase Srs2and promotes recombination by inhibiting the activity of Srs2. Shu complex contains four subunits, Shul, Shu2, Psy3, and Csm2, and only the intact Shu complex has the function to maintain genomic stability. Although Shu complex plays an important role in damage repair, the underlying mechanism is largely unknown.In this work, we have solved the crystal structure of the Psy3-Csm2complex. The structure reveals that although Psy3and Csm2have low sequence identity, they share a similar architecture that closely resembles the ATPase core domain of Rad51. Psy3forms a heterodimer with Csm2mainly through a hydrophobic core. Similar to Rad51, both Psy3and Csm2contain a L2loop that is rich in positively charged amino acids. The L2loops in Psy3and Csm2are DNA binding motifs and confer the DNA binding activity of the Shu complex. As with Rad51, the Shu complex appears to form a nucleoprotein filament by binding nonspecifically to DNA. Through yeast phenotypic assay, we demonstrated that the DNA binding activity of the Shu complex is essential for repair of the methyl methanesulfonate-induced DNA damage. Shu complex may be recruited to regulate the activity of Srs2by binding to the DNA in the damage site.