Structural and functional analysis of DNA binding by the Rad50 catalytic head from Thermotoga maritima [thesis]

Anna Maria Rojowska
Double-strand breaks are among the most deleterious DNA lesions, with a single unrepaired break capable of inducing apoptosis. This highly cytotoxic damage requires a fast and efficient cellular response. DSBs are repaired in two distinct repair pathways: homologous recombination or non-homologous end joining. Both of these repair systems ensure recognition of the damage site, activation of the cell-cycle checkpoint and repair of the lesion. A key player in the DSB repair is the conserved
more » ... the conserved Mre11-Rad50-Nbs1 complex. The enzymatic core of this assembly -Mre11-Rad50 -is highly conserved and found in all domains of life. Eukaryote-specific Nbs1 (Xrs2 in S. cerevisiae) is a signalling subunit transmitting the damage event to the ATM kinase. Rad50 shares homology with the SMC protein family. Its dimer comprises two globular bipartite ATPase domains and two coiled-coil domains. The nuclease Mre11 also forms a dimer and associates with Rad50. This conserved catalytic head is believed to recognize DSB and form a DNA-binding interface. Moreover, the MR complex takes part in the initial processing of DNA ends and signals the lesion to the cell-cycle control machinery activated via Nbs1-ATM interaction. Nbs1 carries Mre11-and ATM-binding motifs and mediates damage recognition with checkpoint activation. The MRN complex is required not only for the repair of mitotic DSBs but also plays a role in meiotic recombination, telomere maintenance and adaptive immune system development. This multifaceted behaviour of the complex lies in its enzymatic activities, as well as DNA bridging and tethering function and activation of ATM-induced repair signalling. This work attempted to characterize molecular principles of DNA recognition by the catalytic part of the bacterial Rad50. To this end, the enzymatic core of T. maritima Rad50 together with an interaction motif of Mre11 was crystallized with AMPPNP and a dsDNA molecule. In the structure, DNA binding is asymmetrical and involves residues on both the globular head of the Rad50 and the root of the coiled-coil domain. Further studies were performed to biochemically characterize the protein-DNA complex and the identified binding sites. The atomic model yielded a number of structural features that were analyzed in vitro and in vivo. Finally, significance of these features together with the current understanding of the DNA binding by the MRN complex are discussed. crosslinks (Waris and Ahsan 2006). These lesions are repaired predominantly by BER and NER systems. However, if this repair fails, the damage imposes stress during replication and can elicit double-strand break response. Double-strand breaks of exogenous source Primary causes of DSBs of extracellular origin are ionizing radiation (IR), UV light and genotoxic agents. The most frequent DNA damage resulting from UV radiation are cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts. These lesions contain covalent linkages between neighbouring thymines or cytosines (Goodsell 2001). UV-induced DNA modifications distort the structure of DNA and hence, are mostly repaired by the NER system. However, unless DSB repair Unrepaired DSBs are among the most deleterious DNA lesions. Therefore, cells have developed a fast and efficient repair system, employing DSBs sensors, repair complexes and a signalling machinery. The two main DSB repair pathways are homologous recombination (HR) and nonhomologous end joining (NHEJ). HR is a highly faithful pathway, in which a non-damaged homologous DNA segment (usually a sister chromatid) governs the repair of the lesion. NHEJ is more error-prone and involves a partial resection of the broken DNA ends followed by ligation. Since NHEJ is not accompanied by a large-scale polymerase gap filling, deletions and sequence alterations often arise in the religated DNA helix. Homologous recombination The process of homologous recombination starts with an extensive resection of the damage site in the 5'-3' direction, resulting in a generation of single-stranded 3' overhangs ( Figure 2 , panel A). Upon sensing the DNA damage, the Mre11-Rad50-Nbs1 (MRN) complex together with the Sae2 nuclease (S. cerevisiae; CtIP in human, Ctp1 in S. pombe) catalyzes removal of a short oligonucleotide from each 5' end. This short-term end processing is followed by a long-term Recent studies shed light on the eukaryotic Mre11 structure (Park et al. 2011; Schiller et al. 2012). Most notably, yeast Mre11 model with Nbs1 peptide revealed first insights into Nbs1 binding by the Mre11 and the involvement of Mre11 structural features in the outbreak of ataxiatelangiectasia-like disease (ATLD). S. pombe Mre11 forms a flexible dimer with asymmetrically bound Nbs1 stabilizing the dimer interface. Furthermore, despite sharing the overall 3D architecture with prokaryotic orthologues, eukaryotic structures possess a large loop insertion at one end of the dimer interface and an additional helix in the capping domain (Schiller et al. 2012). Interestingly, some of the ATLD mutations map to this regions, suggesting its involvement in the DSB-induced signalling. Goodsell, D. S. 2001. The molecular perspective: ultraviolet light and pyrimidine dimers. Oncologist 6 (3):298-9.
doi:10.5282/edoc.17762 fatcat:wsrxz2w7obhcbl7abqfe3yo2d4