Patricia Kane, PhD
Chair and Professor
The central goals of scientists in the Biochemistry and Molecular Biology department are to research fundamental mechanisms in cells and organisms at the molecular level and to train the next generation of scientists. Read More...
The Smc5/6 complex at the crossroads of DNA replication, repair and recombination
Due to the imperfect “steric gate”, DNA polymerase intrinsically mis-incorporates not only mismatched deoxyribonucleosides monophosphates but also ribonucleoside monophosphates (rNMPs) during DNA replication at a rate of 10−7 and 4 × 10−4, respectively (1). To repair these DNA damages it requires specific recognition and excision proteins to remove the damage and create a single-stranded DNA (ssDNA) gap, followed by the DNA polymerase to fill the gap and the DNA ligase to seal the nick. Because ssDNA is highly prone to degradation and abnormal recombination, the question arises how do the cells ensure such ssDNA gaps are well protected and correctly repaired?
One of the evolutionarily conserved Structural Maintenance of Chromosome protein complexes, the Smc5/6 complex, was previously implicated in preventing and/or resolving toxic recombination intermediates in the homologous recombination-mediated DNA repair pathway. The core complex proteins, Smc5 and Smc6, have been shown to have high affinity towards ssDNA. Interestingly, loading of the Smc5/6 complex onto the chromosome is dependent on DNA replication, which suggests it has a functional role in DNA replication. Jie Peng, a Ph.D student in Dr. Wenyi Feng’s laboratory, decided to ask whether the Smc5/6 complex acts as a guardian to prevent extensive ssDNA formation behind the replication fork.
In the August 17 issue of Nucleic Acids Research, using a hypomorphic smc6 mutant Jie showed that in the absence of a functional Smc5/6 complex DNA replication proceeds faster, incorporates more rNMPs, and leaves more ssDNA gaps behind (1). These defects were further exacerbated during replication stress such as lowered nucleotide pools or alkylation damage, and the mutant loses viability. Interestingly, deleting a gene called RNH1, whose function is to excise the mis-incorporated rNMP, can rescue the viability of the smc6 mutant. This observation lent a clue to the researchers that the attempt to repair DNA damage is dangerous for cells with an impaired Smc5/6 complex. They speculated that the repair process is inhibited at a stage following the excision of DNA damage, leaving ssDNA gaps behind for degradation or engagement in toxic recombination intermediate formation. Supporting their hypothesis, Jie found that deleting the equivalent of the RNH1 gene in virtually all other DNA damage repair pathways also rescues the viability of the smc6 mutant.
The Smc5/6 complex is essential for cell survival; however, its role is characterized primarily in the non-essential homologous recombination-mediated DNA repair pathway. This dispute has remained unresolved in the research field for years. This work provided new evidence for a fundamental role of the Smc5/6 complex during replication to prevent nucleotide mis-incorporation, followed by its subsequent role in the repair of these damages. Recently, the Smc5/6 complex has been linked to patients with primordial dwarfism, extreme insulin resistance, and chromosome breakage syndrome with severe lung diseases (3,4). The work by Peng et al. will also contribute to the understanding of the etiology of these diseases.
- Nick McElhinny S, et al. Genome instability due to ribonucleotide incorporation into DNA. Nat. Chem. Biol. 2010;6:774-781.
- Peng J, Feng W. Incision of damaged DNA in the presence of an impaired Smc5/6 complex imperils genome stability. Nucleic Acids Res. 2016. doi:10.1093/nar/gkw720
- Payne F, et al. Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance. J Clin Invest. 2014;124(9):4028-4038.
- Van der Crabben S, et al. Destabilized SMC5/6 complex leads to chromosome breakage syndrome with severe lung disease. J Clin Invest. 2016;126(8):2881-289
New class of p53-reactivating compounds provides novel mechanism to treat cancer
The “Guardian of the Genome,” p53, is a tumor suppressing transcription factor that has long been recognized as perhaps the most important protein in human cancer. Approximately 50% of human cancers harbor mutations in p53, which render the protein inactive and unable to protect the cells from cancerous transformation. Because of this prevalence, p53 has been somewhat of a "White Whale" for those attempting to develop new anti-cancer therapies since it's central role in human cancer was elucidated in the early 90's. However, despite decades of searching, there are no targeted p53 therapies in available in the clinic.
Like many DNA-binding proteins, p53 requires proper coordination of a Zn2+-cofactor for proper folding and function. One of the most common ways the protein is inactivated is by mutational disruption of this interaction. We in the Loh Lab have been studying the details of the interaction between p53 and Zn2+ for over a decade, and in the past few years finally reached a translational breakthrough – along with biological collaborators at the Cancer Institute of New Jersey, we have defined a class of experimental cancer drugs that we call “Zinc Metallochaperones.”
These drugs function by first acting as ionophores: they transport Zn2+ from the serum, where the concentration is relatively high, into the cell, where the concentration is relatively low (Fig. 1). Once inside the cell, the Zinc Metallochaperones act as Zn2+-buffers, raising the intracellular Zn2+ concentration to just the right level to allow defective p53 to bind Zn2+ once again, thereby restoring p53’s structure and function. This mechanism is unprecedented in pharmacology, and represents a fundamentally new approach to target p53 in cancer.
We've demonstrated the effectiveness of this kind of therapy using dozens of pre-clinical models, including purified protein, isogenic cell lines with exogenously expressed p53 mutants, cell lines with diverse genetic backgrounds that endogenously express different p53 mutants, genetic knock-in mouse models, and tumor xenograft mouse models. We are now in the process of conducting a structure activity relationship, and finding compounds that have the potential to go on to human trials.
Reference: Yu X, et al. (2014) Small molecule restoration of wildtype structure and function of mutant p53 using a novel zinc-metallochaperone based mechanism. Oncotarget 5(19):8879–8892.
Blanden AR, et al. (2015) Synthetic metallochaperone ZMC1 rescues mutant p53 conformation by transporting zinc into cells as an ionophore. Mol Pharmacol 87(5):825–831.
Yu X, Vazquez A, Levine AJ, Carpizo DR (2012) Allele-Specific p53 Mutant Reactivation. Cancer Cell 21(5):614–625.
Blanden AR, Yu X, Loh SN, Levine AJ, Carpizo DR Reactivating mutant p53 using small molecules as zinc metallochaperones: awakening a sleeping giant in cancer. Drug Discov Today. doi:10.1016/j.drudis.2015.07.006.
Mechanism of Ant1-induced human disease unraveled by the Chen lab
Mitochondria are the powerhouses of the cell. About 90% of the energy that cells need is produced in the form of ATP by the OXPHOS apparatus on the mitochondrial inner membrane. After its synthesis by the FoF1 ATP-synthase, ATP is exported out of mitochondria via adenine nucleotide translocase (Ant) by exchanging with the cytosolic ADP. Ant is one of the most conserved proteins through evolution and is present in multiple isoforms in different species. In human, the Ant1 isoform is predominantly expressed in skeletal muscle and heart. Several missense mutations in Ant1 (A90D, L98P, D104G, A114P and V289M) cause autosomal dominant progressive external ophthalmoplegia (adPEO), commonly manifested by opthalmoplegia, ptosis and exercise intolerance. Another mutation in Ant1, A123D, causes cardiomyopathy and myopathy in homozygous patients. Several models have been proposed to explain the pathogenic mechanism of these diseases, with most of which suggesting that the pathogenesis results from the altered nucleotide transport activity. The Chen lab has previously observed that missense mutations in the yeast Aac2 protein, mimicking those in human diseases, cause severe mitochondrial damage independent of nucleotide transport. However, the precise mechanism for the Ant1-induced mitochondrial damage remains unknown.
In a recent study by Yaxin Liu, a graduate student in the Chen lab, it was found that the “pathogenic” variants of the Aac2 protein are misfolded. Yaxin developed an in organello assay to examine the properties of Aac2. Isolated mitochondria were incubated under specific conditions before being analyzed by Blue Native PAGE (BN-PAGE). In contrast to the wild type Aac2 protein from wild-type mitochondria that migrated as a monomer on the native gel (see Figure 1), the adPEO-type Aac2 protein formed higher molecular weight bands (>720kD) indicative of protein aggregation. The in organello assay also revealed that the mutant Aac2 is susceptible to proteolytic degradation. Additional experiments by Xiaowen Wang, a research scientist in the Chen lab, showed that cells expressing the mutant variants of Aac2 are hypersensitive to conditions that reduce mitochondrial protein quality control and alter the membrane properties. These data collectively suggested that the mutant Aac2 proteins are misfolded.
How does the misfolded Aac2 damage the mitochondria and eventually induce cell death? To answer this question, Yaxin determined the assembly state of the respiratory complexes, the supercomplexes, and the TIM22 and TIM23 protein translocases on the mitochondrial inner membrane by BN-PAGE. It was found that the structural integrity of these protein complexes is severely affected by the mutant Aac2. The data supported the model that the misfolded Aac2 either induces global proteostatic stress which in turn affects the assembly of the protein complexes on the mitochondrial inner membrane, or that the misfolded Aac2 ectopically interacts with specific protein complexes and affects their stability.
Yaxin’s work has important clinical and biological implications. The data strongly suggested that the human diseases induced by the missense Ant1 variants belong to protein misfolding disorders. It provided an important direction for the development of future therapeutic strategies for the diseases, which should be focused on improving mitochondrial proteostasis rather than stimulating nucleotide transport. More importantly, this piece of work demonstrated that the misfolding of one single protein could have detrimental effects on proteostasis in the membrane. This could have general implications for understanding the physiological outcomes of other misfolded membrane proteins that are involved in ever-increasing number of dominant diseases.
Yaxin is a recipient of a predoctoral fellowship from the American Heart Association. The Chen lab is supported by grants from the National Institute on Aging (NIH).
Reference: Yaxin Liu, Xiaowen Wang and Xin Jie Chen. Misfolding of mutant adenine nucleotide translocase in yeast supports a novel mechanism of Ant1-induced muscle diseases. Mol Biol Cell. 2015 Jun 1;26(11):1985-94.
Using disease-associated mutations to understand the biochemical regulation of a multi-subunit histone methyltransferase complex
Eukaryotic DNA is compacted into chromatin, which must be continually remodeled to allow for DNA dependent processes such as transcription. The basic repeating unit of chromatin, the nucleosome, is composed of an octomer of histone proteins around which 147 base pairs of DNA is wrapped. One way chromatin remodeling is achieved is by posttransitional modification of histones.
In the Cosgrove lab, we are interested in the molecular mechanisms that regulate histone H3 lysine 4 (H3K4) methylation, which is a histone modification required for the inheritance of transcriptionally active states of chromatin. Lysine residues can be mono-, di-, or tri-methylated with each modification leading to distinct functional outcomes. However, the mechanisms that regulate the degree of H3K4 methylation are poorly understood. In humans, the SET1 family of histone methyltransferases are the major implementers of H3K4 methylation. The SET1 family is composed of the mixed-lineage leukemia proteins (MLLs) 1-4 and Setd1a/b that all share the conserved Su(var)E(z)Trx (SET) domain. Mutations in the genes encoding the SET1 family are associated with leukemias, solid tumors, and developmental disorders. All SET1 family members interact with a conserved sub-complex of proteins that include WDR5, RbBP5, ASH2L, and DPY-30 (WRAD). The interaction of a SET1 member with WRAD is known as the core complex and it is required for multiple methylation of H3K4.
We focus on the most well studied human SET1 family member, MLL1, which has served as a paradigm for understanding how the activity of the SET1 family is regulated. We previously found that MLL1 catalyzes H3K4 mono-methylation in the absence of WRAD. However, the conversion to di-methylation is catalyzed by the MLL1 core complex and requires an unknown surface from MLL1 that is distinct from the canonical MLL1 active site. Due to the lack of a high-resolution structure of the MLL1 core complex, the surfaces involved in the formation of the MLL1 core complex and the di-methyltransferase active site remain unknown. Recent genome sequencing studies have identified a number of disease-associated missense mutations that localize to the SET domains of several SET1 family members. Stephen Shinsky, a graduate student in the Cosgrove lab, modeled five MLL-2 missense mutations associated with human Kabuki Syndrome (KS) in the MLL1 core complex. He found that a subset of KS mutations map to a common solvent exposed surface in the MLL1 SET domain (Figure 1). Although all the residues mutated in KS are oriented away from the canonical MLL1 active site, Stephen found that all KS variants displayed loss of H3K4 di-methylation when assembled into the core complex. Stephen discovered that the loss of di-methylation activity is associated with a weakened ability of KS variants to interact with WRAD or the RbBP5-ASH2L heterodimer, which is required for formation of the di-methyltransferase active site (Figure 1). These results suggest that amino acids from this surface of MLL1, which we term the Kabuki interaction surface (KIS), are required for formation of the core complex and di-methyltransferase active site within SET1 family core complexes (Figure 1). Stephen's work was recently published in the Journal of Molecular Biology.
Figure 1. KS missense mutations define a Kabuki interaction surface (KIS) that is crucial for the interaction with the RbBP5/ASH2L heterodimer and for H3K4 di-methylation. (Upper Panel) The two-active site model for multiple lysine methylation by Set1 family complexes. The MLL1 core complex mono-methylates H3K4 at the canonical SET domain active site (indicated by the number 1), which is followed by H3K4 dimethylation at a second active site formed at the interface between MLL and WRAD (indicated by number 2). KIS localized KS mutations in the MLL SET domain weaken the interaction with RbBP5 and ASH2L resulting in the loss of H3K4 di-methylation activity (lower left panel). Alternatively, substitution of R3765 with leucine in the WDR5 interaction (Win) motif destabilizes the interaction between MLL and WRAD thus also preventing formation of the di-methyltransferase active site (lower right panel).
References: Shinsky SA, Hu M, Vought VE, Ng SB, Bamshad MJ, Shendure J and Cosgrove MS. A non-active site SET domain surface crucial for the interaction of MLL1 and the RbBP5-ASH2L heterodimer within MLL family core complexes. Journal of Molecular Biology (2014) 426: 2283-2299
Attack of the Killer Severer - in the Amberg Lab - May 2013
Graduate student Dimitra Aggeli, working in the Amberg Lab, has discovered how to turn the small actin binding protein cofilin into an actin filament destroying machine. Building off the previous work by graduate student Mike Clark, Dimitra has been trying to understand why some of Mike's mutants are hyperactive for actin filament disassembly. Turns out these mutants disrupt one of the two actin binding sites (the secondary filament specific binding site) on cofilin (see Figure 1) thus allowing Dimitra to isolate and study the effects of the primary binding site on actin filament stability.
Dimitra has found that normal cofilin actually promotes actin polymerization and this activity is completely lost by the mutants defective in the secondary binding site. In fact, the mutants rapidly disassemble pre-formed actin filaments. To understand the mechanism of filament destabilization, Dimitra used fluorescently labeled actin filaments to watch the process of filament destruction by the mutants under a microscope. What she found is that the mutants sever (break in the middle) the actin filaments (see the movie below). This is an activity of cofilin that is activated by the protein Aip1p that was discovered by Dr Amberg during his post-doc years. Dimitra's observations not only explain how cofilin severs, an answer long sought by many labs, but they also explain how cofilin severing is regulated and that is by disrupting the secondary binding site. Dimitra is currently writing a paper on her observations. This paper will include data from a previous graduate student of Stephan Wilkens', Erik Kish-Trier, who during a brief stint in the Amberg lab, solved the crystal structure of the three cofilin mutants; one at 1.90Å, one at 1.45Å, and the third at a stunning 1.10Å. Not bad for Erik's first foray into crystallography!
Understanding ε-mediated inhibition of bacterial F-type ATP synthase to develop drugs against Myobacterium tuberculosis in the Duncan Lab
Mycobacterium tuberculosis (MTB) is an infectious pathogen that causes Pulmonary Tuberculosis and kills over one million people every year. It is also a major cause of death in HIV patients. Evolution of extreme drug-resistent strains in MTB poses a serious problem towards its treatment. As a result, there is an ongoing need to develop novel drugs that can effectively fight the resistant strains. The F-type ATP synthase is responsible for energy production in living organisms and was recently identified as a drug target for treatment of MTB. It is a unique rotary motor enzyme that can synthesize as well as hydrolyze ATP depending on its direction of rotation.
The ATP synthase is often called F0F1. It has a membrane embedded F0 complex that transports protons by rotation of its c subunits and an external catalytic F1 complex that is composed of 5 subunits with the stoichiometry of α3β3γδε. The ε subunit is involved in inhibition of many bacterial ATP synthases wherein its C-terminal domain inhibits both ATP synthesis and hydrolysis whereas the N-terminal domain is necessary for functional assembly of the enzyme. Work in the Duncan lab is focused on understanding of ε mediated inhibition of bacterial ATP synthase. Recently, the Duncan lab determined the first high resolution crystal structure of F1 in Escherichia coli (EF1) in an autoinhibited conformation. The structure showed ε's CTD in a highly extended conformation (εx) that engaged in interaction with rotor and stator subunits inside the central cavity of EF1. These interactions of ε with other subunits are responsible for inhibition of the eyzyme.
Naman Shah, a graduate student in the Duncan lab, is studying these interactions of ε within F1 with the help of an optical assay that measures binding and dissociation kinetics of F1/ ε. The binding and dissociation of wild type and different mutants of ε are correlated with their inhibitory effects on the enzyme. A paper describing his results was recently published in the Journal of Biological Chemistry. This work has provided new insights on ε's confirmation when treated with different catalytic-site ligands as well as at different rotation angles of the enzyme. Understanding ε's interaction within F0F1 could lead to development of drugs that specifically target F0F1 in MTB as well as many other pathogenic bacteria. Drugs that can enhance the ε mediated inhibition of F0F1 are expected to kill the pathogen by halting energy metabolism. These drugs should be highly specific towards the pathogenic bacteria as ε's homolog does not have this regulatory role in mitochondrial F0F1.
References: Shah NB, Hutcheon ML, Haarer BK, Duncan TM. (2013) F1-ATPase of Escherichia coli: The ε -inhibited State Forms after ATP Hydrolysis, is Distinct from the ADP-Inhibited State, and Responds Dynamically to Catalytic-Site Ligands. Journal of Biological Chemistry (epub version)
Cingolani G and Duncan TM (2011) Structure of the ATP synthase catalytic complex (F1) from Escherichia coli in an autoinhibited conformation. Nature Structure Molecular Biology 18, 701-707.
New Insight into the Reversible Dissociation of the V-ATPase Revealed by the Wilkens Lab
The vacuolar ATPase (V-ATPase) is a rotary molecular motor enzyme that functions to acidify the lumen of subcellular organelles in all eukaryotic cells. V-ATPase function is involved in a number of fundamental cellular processes including pH/ion homeostasis, endocytosis, vesicular traffic and antigen processing. In the Wilkens Lab, we are studying the structure of the enzyme to gain a more detailed understanding of its mechanism and its unique mode of regulation.
V-ATPase regulation involves a major structural rearrangement wherein the soluble and membrane sectors dissociate from one another (Fig 1). While dissociated, the activity of both V1 (ATPase) and Vo (proton transport) are silenced and a single enzyme subunit (C) is released into the cytosol. The structural basis for this unique mode of regulation is being studied by graduate student, Rebecca Oot, whose work is focused upon subunits that play a role in linking the V1 and Vo sectors during normal enzyme function. Interactions between these subunits, C, H, E, G and aNT, (in color, Fig 1) function to resist the torque generated during rotary catalysis. Interestingly, these interactions are required to be strong enough to maintain the structural integrity of the enzyme during rotary catalysis but must be vulnerable to breakage for regulated enzyme disassembly to occur.
Previously, Rebecca found that subunit EG heterodimer binds subunit C (via Chead) with high affinity (delta G ~-40kJ/mol), an interaction that greatly stabilizes EG in vitro. Recently, Rebecca used purified recombinant subunits and subunit domains for quantitative, biophysical characterization of the interactions between subunit C, EG heterodimer and a soluble subdomain of a membrane embedded subunit, a (aNT). Here, Rebecca found that subunit C (Cfoot domain) forms a ternary binding interface formed from low affinity interactions with another copy of the EG heterodimer and aNT.
We speculate that this interface, composed of Cfoot-EG-aNT, results in a high avidity interaction that may be tunable by environmental signals, such as pH. In the assembled enzyme, this interaction would be equivalent to the product of the individual affinities (low nanomolar Kd). However, breaking of even one of the interactions (EG-aNT or EG-Cfoot) would compromise the integrity of the linkage to the membrane and may facilitate enzyme dissociation. It has been shown that enzyme function is required for regulation to occur and we hypothesize that upon the weakening of this interaction, continued enzyme rotation would serve to physically rip apart the high affinity EG-Chead interaction, leading to loss of subunit C from the enzyme. Importantly, the delta G of association of the high affinity interaction is similar to that of ATP hydrolysis, suggesting that the energetic cost of breaking the interaction could be balanced by hydrolysis of one ATP.
References: Oot RA and Wilkens S. (2012) Subunit Interactions at the V1-Vo Interface in Yeast Vacuolar ATPase. Journal of BIological Chemistry 287: 13396-13406.
Oot RA and Wilkens S. (2010) Domain Characterization and Interaction of the Yeast Vacuolar ATPase Subunit C with the Peripheral Stator Stalk Subunits E and G. Journal of Biological Chemistry 285: 24654-24664.
Research Highlight: Epigenetics of leukemia - research in the Cosgrove lab
Cell identity in multi-cellular organisms is determined in part by factors that regulate gene accessibility within the context of eukaryotic chromatin. Post-translational modifications of histone proteins are central in the establishment of heritable gene expression programs through the regulation of chromatin structure. While a large number of distinct post-translational modifications have been identified in recent years, the molecular mechanisms by which these modifications regulate gene accessibility are not well understood.
We are focusing on identifying the molecular mechanisms for the regulation of histone H3 lysine 4 (H3K4) methylation - an epigenetic mark correlated with transcriptional activation. H3K4 methylation is critical for a number of important biological processes including stem cell differentiation, metazoan development, transcription, and the pathogenesis of cancer. H3K4 can be mono-, di-, or trimethylated, often with distinct functional outcomes. The molecular mechanisms that control the degree of H3K4 methylation are not well understood.
In our laboratory we use molecular, structural and biophysical approaches to investigate the molecular mechanisms of H3K4 methylation by the human Mixed Lineage Leukemia protein-1 (MLL1) core complex. The human MLL1 gene resides at chromosome 11 band q23 and is frequently translocated in aggressive infant and adult acute leukemias. MLL1's H3K4 methyltransferase activity regulates the expression of HOX genes during hematopoiesis and development. MLL1 contains an evolutionarily conserved SET domain that is required for its catalytic activity. MLL1's catalytic activity is regulated through interaction with a conserved core complex of proteins that include WDR5 (WD-repeat protein-5), RbBP5 (retinoblastoma binding protein-5), Ash2L (Absent, small, homeotic dics-2-like) and DPY-30 (Dumpy-30). Together, these proteins form the MLL1 core complex, which is required for the regulation of the degree of H3K4 methylation in eukaryotes. We have recently discovered that the WDR5-RbBP5-Ash2L sub-complex possesses a SET domain-independent methyltransferase activity that catalyzes H3K4 dimethylation within MLL1 core complex. Our results suggest that eukaryotes have evolved a highly intricate system for regulating the degree of H3K4 methylation.
Our long-term goal is to understand the protein-structural features responsible to the enzymatic activity and regulation of H3K4 methylation by the human MLL1 core complex. This information is essential for a broader understanding of the role of H3K4 methylation in the pathways that regulate gene expression in the context of eukaryotic chromatin. In addition, the knowledge gained from work may lead to better diagnostics and the rational design of anti-cancer drugs for the treatment of certain forms of leukemia and other malignancies.
Projects in the lab are available to investigate the structure, enzymology and cell biology of MLL family complexes. Interested Ph.D. applicants should apply at the departmental website. Postdoctoral applicants should contact me directly at: firstname.lastname@example.org
References: Patel, A., Vough, V., Dharmarajan, V., and Cosgrove, M.S. (2011) A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the MLL1 core complex. Journal of Biological Chemistry 286, 3359-3369.
Patel, A., Dharmarajan, V., Vough, V.E., and Cosgrove, M.S. (2009) On the mechanism of multiple lysine methylation by the human Mixed Lineage Leukemia protein-1 (MLL1) core complex. Journal of Biological Chemistry 284, 24242-24256. (Selected as JBC's Paper of the Week).
Research Highlight: An Evolutionarily conserved DNA recombination/repair protein identified in mitochondria
Mitochondrial DNA (mtDNA), encoding integral components of the energy-producing oxidative phosphorylation pathway on the mitochondrial inner membrane, is extremely vulnerable to damages by oxidative, chemical, irradiational and metabolic stresses. However, although mtDNA has been discovered almost 50 years ago, how damaged mtDNA is repaired is poorly understood. Homologous recombination (HR) is a universal molecular process conserved from batcheriophage to human. It has primarily evolved as a mechanism for the repair of double stranded DNA breaks and for the re-initiation of DNA synthesis from collapsed replication forks.
Can the universality of HR be extended to mitochondria? A crucial step to address this question is to demonstrate the presence of DNA recombination machineries in mitochondria. We discovered the yeast MGM101 gene that is essential for mtDNA maintenance in 1993. Other investigators subsequently demonstrated that Mgm101 is a mt-nucleoid protein required for mtDNA repair. Although Mgm101 has also been shown to share some sequence similarities with the recombination protein, Rad52, the biochemical and functional study has been held back in the last decade by the difficulty to produce the recombinant protein. Three years ago, MacMillan Mbantenkhu, a graduate student joined our lab and attempted to express Mgm101 in E. coli. By using the MBP-fusion strategy, Mac was able to produce Mgm101 in large quantities in a soluble form. This important accomplishment permitted him to show that Mgm101 tends to oligomerize in vitro. Xiaowen Wang, a research scientist in the group, then found that like Rad52, Mgm101 preferentially binds to ssDNA. More importantly, she also found that Mgm101 catalyzes single strand DNA annealing even in the presense of the mitochondrial single strand binding protein, Rim1. In collaboration with Stephen Wilkens, we showed that Mgm101 forms distinct oligomeric rings (see Figure, panels A & B) with a diameter of ~200 Å, and highly compressed helical filaments with a pitch of ~50 Å (panels C & D). In the presense of short (panel E) and long (panel F) ssDNA substrates, Mgm101 forms thin and condensed nucleoprotein filaments respectively. When analyzed by sedimentation velocity analytical ultracentrifugation, our collaboratrs, Michael Cosgrove and Anamika Patel, established that each Mgm101 ring contains ~14 subunits. These biochemical and structural properties remarkably resemble those of the Rad52-type recombination proteins such as Redβ, Erf, RecT and Sak from bacteriophages. Together with Jonathan Nardozzi, a post-doctoral fellow in our lab, we have been able to show that several mutations compromising Mgm101 function in vivo also affected Mgm101 oligomerization in vitro. This led to our hypothesis that the Mgm101 rings may act as stores in the mitochondrial nucleoids, which stabilizes the protein by preventing the aggregation of the otherwise highly unstable Mgm101 protomers. Other contributors to this work include Elizabeth Hoffman, a SURF student who helped to show that defect in Mgm101 function affects mtDNA recombination in vivo.
In summary, our finding strongly supports the existence of a recombination system of bacteriophage-origin in mitochondria. This finding could have important implications for better understanding the mechanisms of repair and probably also replication of mtDNA. This piece of work was recently published online in the Journal of Biological Chemistry.
Reference: Mbantenkhu M*, Wang X*, Nardozzi JD, Wilkens S, Hoffman E, Patel A, Costrove MS and Chen XJ. Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination. J Biol Chem 2011; Oct 25 [Epub ahead of print]
Research Highlight: Biosensor development in the Loh lab
The Loh lab designs protein-based switches as platforms for biosensing. Proteins recognize many interesting ligands, but most proteins don't change their structure upon binding. This lack of conformational change makes it difficult to detect the binding event. Therefore, a major challenge is to develop general mechanisms by which ordinary binding proteins can be converted to molecular switches. Meg Stratton, a PhD student in the Loh lab, created a flourescent calcium sensor (calbindin-AFF) based on the calcium-binding protein calbindin D9k. The method she helped develop, called 'alternate frame folding', employs partial amino acid sequence duplication to create two native states of calbindin-AFF (N and N'; top figure). One of the states cannot bind calcium and the other can. Calcium binding drives the conformational change from the former state to the latter (bottom figure). In collaboration with David Eliezer's group at Cornell Weill Medical College, Meg used nuclear magnetic resonance spectroscopy to characterize the solution structures of the calbindin-AFF switch in each conformation. She showed that the duplicated sequences undergo matched order-disorder transitions, so that one segment folds as its twin unfolds (and vice versa), as calbindin-AFF switches from one state to the other. This finding is signficant because the coupled folding-unfolding event is a large, physical change that forms the basis for flourscent and other optical detection in calbindin-AFF and other similarly-engineered protein sensors.
References: Stratton, M.M., McClendon, S., Eliezer, D. and Loh, S.N. (2011). Structural characterization of two alternate conformations in a calbindin D9k-based molecular switch. Biochemistry 50, 5583-5589
Stratton, M.M. & Loh, S.N. (2011). Converting a protein into a switch for biosensing and functional regulation. Protein Sci. 20, 19-29
Stratton, M.M. & Loh, S.N. (2010. On the mechanism of protein fold-switching by a molecular sensor. Proteins: Struct. Funct. Bioinf. 78, 3260-3269
Research Highlight: Bin3 Is Required for Head Development
Several years ago, a graduate student in the Hanes laboratory, Wencheng Zhu, was studying embryonic development in Drosophila melanogaster (fruit fly) and discovered a novel methyltransferase called Bin3 (Bicoid-interacting protein 3; Zhu & Hanes, Gene, 2000). The function of this protein remained a mystery for a long time. Then, a group in Montreal found a human homolog of Bin3 and showed it methylates a small non-coding RNA called 7SK (Jeronimo et al., Mol. Cell, 2007). Now, a postdoc in the Hanes laboratory, Vimi Singh, discovered the biological role of Bin3 (Singh et al., Dev. Biol., 2011). It turns out to play a critical role in translation regulation during embryo development. Without its function, normal head development, at least in the fly, cannot occur.
Specifically, Bin3 appears to methylate 7SK RNA during the process of oogenesis and early blastoderm development. Methylation helps stabilize the 7SK RNA, which in turn acts like a scaffold to allow formation of a protein complex that shuts off translation of caudal mRNA. Bin3 and Bicoid are key members of this complex and work together to ensure that Caudal protein is not expressed in the anterior of the embryo. This allows head and thoracic development to occur (see Figure). In bin3 mutants, Caudal protein is expressed in the head and development is defective leading to embryonic death. Whether human Bin3 plays an analogous role in human embryo development is not yet known, but it would not be surprising if it did.
Reference: Singh, N., Morlock, H. and Hanes, S. D. (2011). The Bin3 RNA methyltransferase interacts with Bicoid and is required for caudal repression in the Drosophila embryo. Dev. Biol. 352:104-115