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Eukaryotic CtMre11 CD and CtMre11 RBD -CtRad50 NBD crystal structures

3. Discussion

3.1 Eukaryotic CtMre11 CD and CtMre11 RBD -CtRad50 NBD crystal structures

complex. The crystal structures of CtMre11CD and CtMre11RBD-CtRad50NBD (CtMRBDRNBD) from C. thermophilum reveal new features of the eukaryotic MR(N) complex and explain the mode of binding between Mre11 and Rad50.

3.1.1 Crystal structure of the catalytic domain of CtMre11

For structural information about the C. thermophilum MR(N) complex, the structure of the catalytic domain of CtMre11 was determined. In the CtMre11CD structure the complete eukaryotic insertion loops were modeled into the electron density (Chapter 2.1, Figure 1). The overall architecture is similar to the Nbs1-bound Schizosaccharomyces pombe Mre11CD (SpMre11CD-Nbs1) crystal structure. However, the CtMre11CD structure is even more compact due to a slight movement of the capping domain towards the nuclease active site. Interestingly, the eukaryotic specific insertion loops are ordered in a similar fashion like in SpMre11CD-Nbs1 (Seifert et al. 2015). In the SpMre11CD-Nbs1 structure the Nbs1 peptide interacts with and stabilizes the eukaryotic insertion loops (Schiller et al. 2012). In the CtMre11CD the Nbs1-binding site of the SpMre11CD-Nbs1 structure is occupied by symmetry related molecules and the ordering of these insertion loops could additionally be stabilized by crystal packing. The dimer interface is characterized by mainly hydrophobic interactions between helices Į2 and Į3 of each protomer, hydrogen bonds with Arg66 as well as interactions between the eukaryotic insertion loops. The larger dimer interface, compared to archaeal Mre11, indicates a stronger interaction between the CtMre11 protomers (Seifert et al. 2015). The recently found MRE11 mutation in a PMA (progressive myoclonic ataxia) patient leads to a substitution of Ala for Val at position 47, which could disturb the interaction between Nbs1 and Mre11 (Miyamoto et al. 2014). The amino acid (aa) substitution is located in helix Į1 where in the SpMre11CD-Nbs1 crystal structure an interaction between this helix and the Nbs1 fragment has been identified (PDB code 4FBK) (Schiller et al. 2012).

However, the interacting Arg518 is replaced by Leu in human Nbs1. Additionally, cells from this PMA patient show decreased MRN expression levels (Miyamoto et al. 2014).

149 Interestingly, the human Mre11CD crystal structure represents a different conformation of the dimer interface. Thereby, the helices Į2 and Į3 do not form the characteristic hydrophobic dimerization domain, but the dimer is stabilized by a disulfide bond between Cys146 of each protomer (Park et al. 2011). Further, the interaction between the eukaryotic insertion loops from each protomer is disturbed compared to the CtMre11CD structure (Seifert et al. 2015). It is also unclear how Nbs1 is able to bridge the dimer interface in the conformation of the human Mre11CD structure.

Very recently, it was reported that mutations in the MRE11 yeast gene suppress the effect of CtIP (Sae2) deletion on DNA damage repair in vivo. One mutation is located in the eukaryotic specific insertion loop and probably decreases the interaction between Mre11 and Nbs1 (Xrs2) (Chen et al. 2015). Thereby, the mutated Pro110 corresponds to Pro110 in CtMre11 and Pro119 in SpMre11, which forms a hydrogen bond with Lys526from SpNbs1 (Schiller et al. 2012). Since Nbs1 stimulates DNA unwinding and DNA binding of MRN, the reported mutation might suppress these effects (Paull and Gellert 1999, Trujillo et al. 2003). Interestingly, the mutation and the resulting suppression of the sae2ǻ phenotype are independent of the Mre11 nuclease activity. Additional experiments indicate that independent of the Mre11 nuclease, CtIP is important for the removal of MRN (MRX) from DSBs (Chen et al. 2015). However, in vitro experiments showed that CtIP (Sae2) promotes the endonucleolytic cut by MRN (Cannavo and Cejka 2014).

Together, these studies reveal two functions of the CtIP(Sae2)-MRN interaction. One is the initiation of resection and the other function is the removal of MRN from DSBs.

Considering the fact that yeast CtIP (Sae2) itself shows endonuclease activity in vitro, more detailed research is needed to unravel this MRN-CtIP pathway (Lengsfeld et al.

2007).

3.1.2 Crystal structure of dimeric CtMre11RBD-CtRad50NBD

The crystal structure of ATPȖS bound CtMre11RBD-CtRad50NBD (CtMRBDRNBD) reveals interesting features of the eukaryotic MR(N) complex. The CtRad50NBD structure represents the characteristic overall shape of Rad50NBD known from other prokaryotic crystal structures (Hopfner et al. 2000b, Lammens et al. 2011, Williams et al. 2011). It is characterized by the globular domain, which consists of interacting Rad50 N- and C-terminus, and the truncated coiled-coil (CC) domain. The non-hydrolysable ATP analog

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ATPȖS and the magnesium ion are bound between the conserved Walker A, Walker B and signature motifs (Chapter 2.2, Figure 1). Based on the crystal structure of the CtRad50NBD dimer, six eukaryotic insertions are recognized in comparison to prokaryotic Rad50NBD (Chapter 2.2, Figure 2).

Another interesting characteristic of the CtRad50NBD structure is a very sulfur rich cluster in close proximity to the ATPase domain. This cluster contains four methionines (Met166, Met1194, Met1201 and Met1203) and one cysteine (Cys1207). Whether the oxidation state of these residues plays an important role for the DNA repair under oxidative stress conditions has to be investigated intensively. Since ATM gets activated by oxidative stress, the same could be true for the MRN complex (Paull 2015). From these five residues the Met1194, Met1203, Cys1207 are conserved in eukaryotes and in some eukaryotes Met166 and Met1201 are replaced by other hydrophobic amino acids.

Thus, the cluster forms a very hydrophobic area, which also could play a more structural than a regulatory role.

Figure 10: Crystal structure of CtMRBD-RNBD protomer (MRBD: blue; RNBD: light orange) with highlighted rad50S mutations (red). ATPȖS (magenta/gray) and the magnesium ion (green) are depicted.

Previously, the description of rad50S (separation-of-function) mutations in yeast revealed an impaired meiotic recombination phenotype but show no survival effect under DNA damaging conditions. The rad50S mutations are located in lobe I and are mostly found in

151 the surface exposed ȕ-sheets ȕ1, ȕ2, ȕ4 and ȕ5. These mutations consist of Lys6Glu, Ser14Pro, Arg20Met, Glu21Lys, Val63Glu, Gln79Lys, Lys81Ile, Asn97Asp and Gln99Lys in yeast which correspond to residues Lys6, Ser14, P20, Glu21, Ala64, Gln80, Lys82, Asn98 and Gln100 in CtRad50 (Figure 10) (Alani et al. 1990). Whether the mutated residues are necessary for the interaction with meiotic recombination factors or whether they play a regulatory function in MRN, has to be investigated in future studies.

Also the partly substitution of hydrophobic residues by polar or charged amino acids and vice versa, makes it more difficult to predict a structural function of the rad50S mutations.

3.1.3 Comparison between CtRad50NBD and prokaryotic Rad50NBD structures Comparison with dimerized prokaryotic Rad50NBD crystal structures reveals six insertions in the eukaryotic CtRad50NBD. These insertions and the elongated C-terminus of CtRad50NBD enlarge the surface exposed area of the protein. Insertion I is located in ȕ-sheet ȕ1 and consists of amino acids 17–19. Insertion II is located near the CtRad50NBD dimer interface. It is close to the ATP binding Walker A domain and its conformation might be regulated by the nucleotide state. Previous results from archaeal MR show that this region (Leu51–Arg67 in Pyrococcus furiosus Rad50; corresponding to Leu55–Lys75 in CtRad50) undergoes structural rearrangements upon ATP binding (Williams et al.

2011). Insertion III enlarges ȕ-sheet ȕ6 and thereby especially residues Arg105, Lys108, Arg109 increase the positively charged area in the dimer groove. Insertion IV is the largest eukaryotic insertion. It forms a large hairpin structure that consists of ȕ-sheets ȕ8 and ȕ9. It is located adjacent to the Rad50 CCs and contains a relatively conserved YNYR motif afterwards. Whether insertion IV plays a role in the CC orientation or the YNYR motif is functionally important, has to be analyzed. Additionally, insertion V elongates helix ĮJ by five residues and is located in the same area like Insertion VI and the elongated Rad50 C-terminus.

3.1.4 The C-terminal CtMre11 Rad50-binding domain

At the beginning of this work the eukaryotic mode of binding between Mre11 and Rad50 was unclear. Prokaryotic structures of the Rad50NBD bound to the Mre11RBD reported an

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interaction between the base of the Rad50 CCs and two or three helices in the Mre11 C-terminal region. The structure of CtMRBDRNBD revealed a large C-terminal Mre11RBD consisting of five Į-helices. This domain interacts with the CtRad50 CCs and the interactions are facilitated by mainly hydrophobic residues. The C-terminus of Mre11RBD points towards the globular domain of the dimerized CtRad50NBD (Chapter 2.2, Figure 1).

Interestingly, a mutation in this Mre11RBD has been found in an ATLD patient, which indicates functional importance of the RBD (Delia et al. 2004). The conformation of the Mre11RBD might also be important for the function of MRN, since the Mre11 C-terminus is able to interact with DNA and is playing an important role in meiotic recombination (Furuse et al. 1998, Usui et al. 1998, Bhattacharyya et al. 2008). A sequence alignment of the Mre11RBD reveals less conservation among eukaryotes, which makes it more difficult to predict functional important residues (Chapter 2.2, supplementary Figure S2).

According to the Mre11RBD-Rad50NBD (MRBDRNBD) crystal structures from P. furiosus and Methanocaldococcus jannaschii, the first Į-helix in the RBD is not present in P.

furiosus MRBDRNBD. Additionally, in the Thermotoga maritima (Tm) and M. jannaschii (Mj) MRNBD crystal structures the CCs are disturbed at the position where Mre11 interacts with Rad50 (Lim et al. 2011, Möckel et al. 2012). However, the C.

thermophilum and P. furiosus MRBDRNBD structures reveal continuous Į-helices in this region (Williams et al. 2011) (Chapter 2.2, Figure 2).