Tethering on the brink: the evolutionarily conserved Mre11–Rad50 complex

doi:10.1016/S0968-0004(02)02144-8

Trends in Biochemical Sciences

Volume 27, Issue 8, 1 August 2002, Pages 410-418

https://doi.org/10.1016/S0968-0004(02)02144-8 Get rights and content

Abstract

Mre11–Rad50 (MR) proteins are encoded by bacteriophage, eubacterial, archeabacterial and eukaryotic genomes, and form a complex with a remarkable protein architecture. This complex is capable of tethering the ends of DNA molecules, possesses a variety of DNA nuclease, helicase, ATPase and annealing activities, and performs a wide range of functions within cells. It is required for meiotic recombination, double-strand break repair, processing of mis-folded DNA structures and maintaining telomere length. This article reviews current knowledge of the structure and enzymatic activities of the MR complex and attempts to integrate biochemical information with the roles of the protein in a cell.

Section snippets

The MR family of proteins

The MR family of proteins includes gp47 (M) and gp46 (R) of bacteriophage T4, SbcD (M) and SbcC (R) of Escherichia coli, and Mre11 (M) and Rad50 (R) of eukaryotes and archaebacteria 2., 3., 4.. These MR proteins share the same organizational arrangement (Fig. 2). Amino acid sequence comparisons reveal the presence of several conserved amino acid motifs within the M subunit that are also found in a variety of nucleases and other phosphoesterases 2., 3.. In the R subunit of MR complexes, Walker A

Genetic and cellular observations

In Saccharomyces cerevisiae, Mre11 and Rad50 interact with each other and with a third polypeptide, Xrs2, to form the Mre11–Rad50–Xrs2 (scMRX) complex 8., 9., 10.. Null mutants of rad50, mre11 and xrs2 are sensitive to the damage inflicted by ionizing radiation and methylmethane sulfonate (MMS) ([11] and refs therein) but, surprisingly, have a hyper-recombination phenotype for mitotic interchromosomal recombination (Box 1). In addition, mutations in rad50 and mre11 result in elevated levels of

Biochemical observations

The earliest physical demonstration of the function of an MR complex came from studies with bacteriophage T4. Cellular fractionation and analytical centrifugation experiments indicated that gp46 and gp47 (T4 MR) comprise a membrane-associated nuclease complex that acts to generate single-strand regions at DNA nicks and duplex ends 26., 27..

The SbcD (M) and SbcC (R) polypeptides of E. coli were shown to interact and form a large (1.2 mDa) complex that functions as an ATP-dependent, 3′ to 5′

Crystal structures and direct visualization of MR complexes

X-ray crystallographic and microscopic analyses of the structures of various MR complexes are revealing fascinating insights into the molecular architecture, structural conservation and functional mechanics of these proteins. The P. furiosus MR complex (pfMR) has been purified and shown to possess similar exo–endonuclease activities to SbcCD [4]. In addition, the crystal structures of crucial parts of the pfMre11 and pfRad50 polypeptides have been solved 38., 39.. When the N- and C-terminal

Conclusion

MR complexes play a vital role in many processes involved in the metabolism of DNA ends (e.g. DSBR, maintenance of telomere length, hairpin DNA processing, and sensing and signalling the presence of DNA ends to cell-cycle regulatory apparatus). Via these processes, MR complexes act to maintain genome stability. They are nucleases with a complex molecular architecture, reminiscent of the SMC proteins. Such a structure suggests that the proteins play a structural role in addition to their

Acknowledgements

We thank Andrew Coulson for generating the sequence alignments shown in Fig. 2. We thank Karl-Peter Hopfner, Roland Kanaar and Claire Wyman for critical reading of the manuscript. Our work is supported by the Medical Research Council (UK).

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Nijmegen breakage syndrome 1 (NBS1) protein is a core member of the MRE11-RAD50-NBS1 (MRN) complex that plays a crucial role in DNA damage sensing and repair in plants. Here we report that NBS1 from moss Physcomitrium patens reduces oxidative damage by lowering the cellular ROS in addition to its known role in oxidative DNA damage recovery. Real-time transcript analysis showed up-regulation of the PpNBS1 transcript under different stress conditions. Bacterial cells showed better cell survivability upon over-expressing PpNBS1 protein as compared to untransformed cells. Likewise, overexpression of PpNBS1 in tobacco plants provides improved protection against oxidative damage and exhibited a lesser amount of ROS upon exposure to oxidative stress. Moreover, PpNBS1 contributes to the antioxidant defense mechanism by positively regulating the expression of the antioxidant genes under stress conditions in transgenic tobacco plants. PpNBS1 expressing transgenic tobacco plants resulted in lesser membrane damage, lower lipid peroxidation level, and higher chlorophyll content under stress conditions. Taken together, we conclude in addition to its known role as DNA damage sensor, PpNBS1 also plays a definite role in oxidative stress mitigation by minimizing ROS accumulation in the cell.
Size-dependent antirecombinogenic effect of short spacers on palindrome recombinogenicity
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Palindromic sequences in DNA can instigate genetic recombination and genome instability, which can result in devastating conditions such as the Emmanuel syndrome. Palindrome recombinogenicity increases with its size and sequence similarity between palindrome arms, while quasipalindromes with long spacers are less recombinogenic. However, the minimal spacer length, which could reduce or abolish palindrome recombinogenicity in the eukaryotic genome, was never determined. Therefore, we constructed a series of palindromes containing spacers of lengths ranging from 0 (perfect palindrome) to 10 bp and tested their recombinogenicity in yeast Saccharomyces cerevisiae. We found that a 7 bp spacer significantly reduces 126 bp palindrome recombinogenicity, while a 10 bp spacer completely stabilizes palindromes up to 150 bp long. Additionally, we showed that palindrome stimulated recombination rate is not dependent on Mus81 and Yen1 endonucleases. We also compared the recombinogenicity of a perfect 126 bp palindrome and a corresponding quasipalindrome consisting of the same palindrome arms with a stabilising 10 bp spacer in sgs1Δ and rad27Δ backgrounds, since both Sgs1 helicase and Rad27 endonuclease are implicated in preventing hairpin formation at palindromic sequences during replication.
Functional evaluation of the C-terminal region of bacteriophage T4 Rad50
2020, Biochemical and Biophysical Research Communications
Bacteriophage T4 encodes orthologs of the proteins Rad50 (gp46) and Mre11 (gp47), which form a heterotetrameric complex (MR) that participates in the processing of DNA ends for recombination-dependent DNA repair. Crystal and high-resolution cryo-EM structures of Rad50 have revealed DNA binding sites near the dimer interface of Rad50 opposite of Mre11, and near the base of the coiled-coils that extend out from the globular head domain. An analysis of T4-Rad50 using sequenced-based algorithms to identify DNA binding residues predicts that a conserved region of positively charged residues near the C-terminus, distal to the observed binding sites, interacts with DNA. Mutant proteins were generated to test this prediction and their enzymatic and DNA binding activities were evaluated. Consistent with the predictions, the Rad50 C-terminal mutants had reduced affinity for DNA as measured by Rad50 equilibrium DNA binding assays and an increased Km-DNA as determined in MR complex nuclease assays. Moreover, the allosteric activation of ATP hydrolysis activity due to DNA binding was substantially reduced, suggesting that these residues may be involved in the communication between the DNA and ATP binding sites.
Phage T4 endonuclease SegD that is similar to group I intron endonucleases does not initiate homing of its own gene
2018, Virology
Homing endonucleases are a group of site-specific endonucleases that initiate homing, a nonreciprocal transfer of its own gene into a new allele lacking this gene. This work describes a novel phage T4 endonuclease, SegD, which is homologous to the GIY-YIG family of homing endonucleases. Like other T4 homing endonucleases SegD recognizes an extended, 16 bp long, site, cleaves it asymmetrically to form 3′-protruding ends and digests both unmodified DNA and modified T-even phage DNA with similar efficiencies. Surprisingly, we revealed that SegD cleavage site was identical in the genomes of segD^– and segD⁺ phages. We found that segD gene was expressed during the T4 developmental cycle. Nevertheless, endonuclease SegD was not able to initiate homing of its own gene as well as genetic recombination between phages in its site inserted into the rII locus.
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Citation Excerpt :
It is interesting to notice that this length is highly conserved among bacterial and eukaryotic SMC proteins (Melby et al., 1998). In contrast, the length of the coiled-coil arms of “SMC-like” Rad50/SbcC proteins is variable (Connelly and Leach, 2002), suggesting that the length conservation must have a mechanistic ground that is uniquely associated with the action of SMC proteins. One of the plausible ideas would be that the coiled-coil length is directly related to the physical properties of DNA.
Condensins are large protein complexes that play a central role in chromosome organization and segregation in the three domains of life. They display highly characteristic, rod-shaped structures with SMC (structural maintenance of chromosomes) ATPases as their core subunits and organize large-scale chromosome structure through active mechanisms. Most eukaryotic species have two distinct condensin complexes whose balanced usage is adapted flexibly to different organisms and cell types. Studies of bacterial condensins provide deep insights into the fundamental mechanisms of chromosome segregation. This Review surveys both conserved features and rich variations of condensin-based chromosome organization and discusses their evolutionary implications.
Single substitution in bacteriophage T4 RNase H alters the ratio between its exo- and endonuclease activities
2015, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
The article describes substitutions in bacteriophage T4 RNase H which provide so called das-effect. Phage T4 DNA arrest suppression (das) mutations have been described to be capable of partially suppressing the phage DNA arrest phenotype caused by a dysfunction in genes 46 and/or 47 (also known as Mre11/Rad50 complex). Genetic mapping of das13 (one of the das mutations) has shown it to be in the region of the rnh gene encoding RNase H. Here we report that Das13 mutant of RNase H has substitutions of valine 43 and leucine 242 with isoleucines. To investigate the influence of these mutations on RNase H nuclease properties we have designed a novel in vitro assay that allows us to separate and quantify exo- or endonuclease activities of flap endonuclease. The nuclease assay in vitro showed that V43I substitution increased the ratio between exonuclease/endonuclease activities of RNase H whereas L242I substitution did not affect the nuclease activity of RNase H in vitro. However, both mutations were necessary for the full das effect in vivo. Molecular modelling of the nuclease structure suggests that V43I substitution may lead to disposition of H4 helix, responsible for the interaction with the first base pairs of 5′end of branched DNA. These structural changes may affect unwinding of the first base pairs of gapped or nicked DNA generating a short flap and therefore may stabilize the DNA-enzyme complex. L242I substitution did not affect the structure of RNase H and its role in providing das-effect remains unclear.

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Trends in Biochemical Sciences

ReviewTethering on the brink: the evolutionarily conserved Mre11–Rad50 complex

Abstract

Section snippets

The MR family of proteins

Genetic and cellular observations

Biochemical observations

Crystal structures and direct visualization of MR complexes

Conclusion

Acknowledgements

Cell

J. Biol. Chem.

Genetics

Curr. Biol.

J. Mol. Biol.

J. Virol.

Genes Dev.

Mol. Cell

Nucleic Acids Res.

Cell

J. Biol. Chem.

J. Cell Biol.

EMBO J.

Recombination at double-strand breaks and DNA ends: conserved mechanisms from phage to humans

Mol. Cell

Mutational analysis of a Ser/Thr phosphatase

J. Biol. Chem.

Structural and functional similarities between the SbcCD proteins of Escherichia coli and the RAD50 and MRE11 (RAD32) recombination and repair proteins of yeast

Mol. Microbiol.

Mre11 and Rad50 from Pyrococcus furiosus: cloning and biochemical characterization reveal an evolutionarily conserved multiprotein machine

J. Bacteriol.

hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks

Mol. Cell. Biol.

Cell-cycle regulation of mammalian DNA double-strand break repair

Am. J. Hum. Genet.

A DNA damage response pathway controlled by Tel1 and the Mre11 complex

Mol. Cell

Interaction of Mre11 and Rad50: two proteins required for DNA repair and meiosis-specific double-strand break formation in Saccharomyces cerevisiae

Genetics

The yeast RAD50 gene encodes a predicted 153-kD protein containing a purine nucleotide-binding domain and two large heptad-repeat regions

Genetics

Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants

Nat. Genet.

Review
Tethering on the brink: the evolutionarily conserved Mre11–Rad50 complex