In prokaryotes and archaea the MRN complex consists only of the Mre11 dimer and the Rad50 dimer157. Despite little sequence conservation, the structure of the Mre11 N-terminus, and the general complex assembly is quite conserved amongst species116. However, in contrast to the eukaryotic complex, E. colicells do not require MR to be viable264. In E. coli the homologs of Mre11 and Rad50 are called SbcD and SbcC, respectively. For clarity reasons, theE.colicomplex will be named EcMR throughout the text.
EcMR has similar enzymatic activities to eukaryotic MRN. 30-50 exonuclease activity on dsDNA ends and endonuclease activity on closed ssDNA was observed for EcMR 265,266. In contrast to the eukaryotic complex, the EcMR dsDNA exonuclease activity is ATP-dependent. Cleavage of ssDNA only requires Mre11 and is ATP and Rad50 independent265. EcMR is also able to cleave hairpins, preferably at the 50 end of the bubble near the transition site from loop to double-strand266,267. This cleavage is dependent on the binding of ATP but not its hydrolysis.
The resulting products carry a 50 phosphate group and a 30 OH group267. Interestingly, one study found that EcMR cleaves hairpins, dsDNA and also cruciforms in a plasmid in steps of
∼10 bp268.
Similar to the eukaryotic MRN complex, EcMR was shown to incise blocked DNA ends with 20-28 nucleotides distance to the block269. Cleavage of a blocked DNA end is a relevant in vivo activity and helps to clear abortive topoisomerases and one study reported elevated gyrase-DNA levels in E.coli cells lacking the MR complex (see Figure 15)264.
DSB repair inE.colirelies mainly on the RecBCD pathway and to a lesser extend on the RecFOR pathway (see Section 1.2.2). However, one study found, that E.coli cells are less viable when EcMR is deleted in a strain in which DSBs are introduced by a DNA-methylation dependent endonuclease, which might point to a role in DSB repair depending on the type of DSB270. It should be noted, that the role of MR possibly differs in different bacterial cells. For instance, in Bacillus subtilis, MR deficient cells show slightly increased MMC sensitivity and271 and in Deinococcus radiodurans deletion of MR causes increased susceptibility toγ-radiation272. In E.coli, Rad50 seems to localise to replication forks, while Mre11 was distributed throughout the cell273. EcMR is partly regulated by RpoS, a factor that responds to different types of cellular stress, e.g. starvation, high/low pH or DNA damage274.EcMR expression increases RpoS dependent during the post exponential phase in rich medium or was constitutively upregulated during growth on minimal medium273.
What is the function ofEcMR if it is not required for the viability of cells? Several studies find a connection between genetic stability and EcMR. Early experiments indicated that Rad50 is involved in the processing of palindromic DNA structures and interferes with the replication of a palindrome containing Phage λ275. Later it was shown, thatEcMR activity on a palindrome is replication dependent and that a two-ended DSB is created after the replication fork passed. The repair of this DSB requires proteins from the RecBCD pathway, the replication restart protein PriA and the Holliday junction processing enzymes RuvABC and RecG276. Palindromes and
Figure 15: Main enzymatic activities of EcMR.The four main nucleolytic activities ofEcMR include ssDNA endonuclease activity, 30-50 exonuclease activity, hairpin opening activity and dsDNA endonuclease activity on blocked DNA ends.
interrupted palindromes (also spaced inverted repeats) in DNA are places of increased genomic instability and their ability to form hairpin and cruciform structures can result in the alteration in DNA sequence e.g. deletions277.EcMR seems to play an important role in maintaining the genome integrity, since inverted chromosomal duplications were found in EcMR RecA mutant strains of E.coli278.
Similar to palindromes, triplet repeats are a cause of DNA instability and different human diseases279. Triplet repeats are able to form pseudo-hairpins and EcMR is able to cleave the pseudo-hairpin in a similar fashion as a regular hairpin on the loop/duplex-DNA transition region. The ability to cleave these pseudo-hairpins prevents E.coli from amplification of triplet repeats and thus a higher degree of DNA instability266. This has also been shown on a cellular level, where deletion ofEcMR was necessary to generate triplet repeat expansion280. Similarly, propagation of a plasmid with (TGG)24 present on the leading strand is severely impaired, however, deletion of EcMR restores propagation inE.coli281.
Additionally, EcMR might be involved in the final steps of DNA replication. To complete DNA replication in bacteria, the two generated DNA strands have to be merged at theterregion. This process seems to dependend on EcMR and ExoI. Mutations of EcMR and ExoI gives viable cells, however, close inspections of the ter region shows DNA amplifications. Joining of the DNA ends is also dependent on RecA and RecBCD and EcMR is possibly required to cleave an over replicated intermediate282.
Analysis of EcMR with scanning force microscopy (SFM) indicated a mainly heterotetrameric form (M2R2, 73%) in which the coiled coil apexes present the main interaction interface283. Heterodimers have been observed to a lesser extend (21%) as well as multimers (4%). The overall contour length from apex to head domain was 39 nm, albeit heterogeneity in the length
complexes of human and yeast protein (heterotetramers mainly joined at the heads with or without zinc hook engagement and a higher amount of multimers). However, it should be noted, that no ATP or ATPγS was used in this study283. Since ATP leads to the engagement of the head complex181 this might be the reason for the observed dimeric head structures ofEcMR.
The only structural information available on EcMR before this thesis came from a crystal structure of the Mre11 dimer with the residues 1-340 (nuclease and capping domain)174. This crystal structure shows that the active site is very similar to the structures of PfMre11 and TmMre11. Differences between the structures could be found in other parts e.g. the angle between the two nuclease domains. However, since Rad50 is not present in these structures the differences might be attributed to the missing ATPase. The mutation of any of the active site residues (D8, H10, D48, N83, H84, H184, H222, H224) results in a nuclease dead protein. Like the eukaryotic Mre11, the nuclease is clearly dependent on Mn2+ ions, since only the addition of manganese results in DNA degradation by Mre11174.