Alba Guarné

Crystal structure of the Xrcc4 DNA repair protein and implications for end joining

XRCC4 is essential for carrying out non‐homologous DNA end joining (NHEJ) in all eukaryotes and, in particular, V(D)J recombination in vertebrates. Xrcc4 protein forms a complex with DNA ligase IV that rejoins two DNA ends in the last step of V(D)J recombination and NHEJ to repair double strand breaks. XRCC4‐defective cells are extremely sensitive to ionizing radiation, and disruption of the XRCC4 gene results in embryonic lethality in mice. Here we report the crystal structure of a functional fragment of Xrcc4 at 2.7 Å resolution. Xrcc4 protein forms a strikingly elongated dumb‐bell‐like tetramer. Each of the N‐terminal globular head domains consists of a β‐sandwich and a potentially DNA‐binding helix– turn–helix motif. The C‐terminal stalk comprising a single α‐helix >120 Å in length is partly incorporated into a four‐helix bundle in the Xrcc4 tetramer and partly involved in interacting with ligase IV. The Xrcc4 structure suggests a possible mode of coupling ligase IV association with DNA binding for effective ligation of DNA ends.

Structure of the MutL C‐terminal domain: a model of intact MutL and its roles in mismatch repair

MutL assists the mismatch recognition protein MutS to initiate and coordinate mismatch repair in species ranging from bacteria to humans. The MutL N‐terminal ATPase domain is highly conserved, but the C‐terminal region shares little sequence similarity among MutL homologs. We report here the crystal structure of the Escherichia coli MutL C‐terminal dimerization domain and the likelihood of its conservation among MutL homologs. A 100‐residue proline‐rich linker between the ATPase and dimerization domains, which generates a large central cavity in MutL dimers, tolerates sequence substitutions and deletions of one‐third of its length with no functional consequences in vivo or in vitro. Along the surface of the central cavity, residues essential for DNA binding are located in both the N‐ and C‐terminal domains. Each domain of MutL interacts with UvrD helicase and is required for activating the helicase activity. The DNA‐binding capacity of MutL is correlated with the level of UvrD activation. A model of how MutL utilizes its ATPase and DNA‐binding activities to mediate mismatch‐dependent activation of MutH endonuclease and UvrD helicase is proposed.

Structure and function of the N‐terminal 40 kDa fragment of human PMS2: a monomeric GHL ATPase

Human MutLα, a heterodimer of hMLH1 and hPMS2, is essential for DNA mismatch repair. Inactivation of the hmlh1 or hpms2 genes by mutation or epigenesis causes genomic instability and a predisposition to hereditary non‐polyposis cancer. We report here the X‐ray crystal structures of the conserved N‐terminal 40 kDa fragment of hPMS2, NhPMS2, and its complexes with ATPγS and ADP at 1.95, 2.7 and 2.7 Å resolution, respectively. The NhPMS2 structures closely resemble the ATPase fragment of Escherichia coli MutL, which coordinates protein–protein interactions in mismatch repair by undergoing structural transformation upon binding of ATP. Unlike the E.coli MutL, whose ATPase activity requires protein dimerization, the monomeric form of NhPMS2 is active both in ATP hydrolysis and DNA binding. NhPMS2 is the first example of a GHL ATPase active as a monomer, suggesting that its activity may be modulated by hMLH1 in MutLα, and vice versa. The potential heterodimer interface revealed by crystallography provides a mutagenesis target for functional studies of MutLα.

Structure of the endonuclease domain of MutL: unlicensed to cut

DNA mismatch repair corrects errors that have escaped polymerase proofreading, increasing replication fidelity 100- to 1000-fold in organisms ranging from bacteria to humans. The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonuclease domain of Bacillus subtilis MutL. The structure is organized in dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Additional conserved motifs cluster around the lever and define a Zn(2+)-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mechanism to prevent undesired nicking of newly replicated DNA and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL. The structure also provides a molecular framework to propose and test additional roles of MutL in mismatch repair.

Acyldepsipeptide Antibiotics Induce the Formation of a Structured Axial Channel in ClpP: A Model for the ClpX/ClpA-Bound State of ClpP

In ClpXP and ClpAP complexes, ClpA and ClpX use the energy of ATP hydrolysis to unfold proteins and translocate them into the self-compartmentalized ClpP protease. ClpP requires the ATPases to degrade folded or unfolded substrates, but binding of acyldepsipeptide antibiotics (ADEPs) to ClpP bypasses this requirement with unfolded proteins. We present the crystal structure of Escherichia coli ClpP bound to ADEP1 and report the structural changes underlying ClpP activation. ADEP1 binds in the hydrophobic groove that serves as the primary docking site for ClpP ATPases. Binding of ADEP1 locks the N-terminal loops of ClpP in a β-hairpin conformation, generating a stable pore through which extended polypeptides can be threaded. This structure serves as a model for ClpP in the holoenzyme ClpAP and ClpXP complexes and provides critical information to further develop this class of antibiotics.

ClpP: a structurally dynamic protease regulated by AAA+ proteins.

Proteolysis is an important process for many aspects of bacterial physiology. Clp proteases carry out a large proportion of protein degradation in bacteria. These enzymes assemble in complexes that combine the protease ClpP and the unfoldase, ClpA or ClpX. ClpP oligomerizes as two stacked heptameric rings enclosing a central chamber containing the proteolytic sites. ClpX and ClpA assemble into hexameric rings that bind both axial surfaces of the ClpP tetradecamer forming a barrel-like complex. ClpP requires association with ClpA or ClpX to unfold and thread protein substrates through the axial pore into the inner chamber where degradation occurs. A gating mechanism regulated by the ATPase exists at the entry of the ClpP axial pore and involves the N-terminal regions of the ClpP protomers. These gating motifs are located at the axial regions of the tetradecamer but in most crystal structures they are not visible. We also lack structural information about the ClpAP or ClpXP complexes. Therefore, the structural details of how the axial gate in ClpP is regulated by the ATPases are unknown. Here, we review our current understanding of the conformational changes that ClpA or ClpX induce in ClpP to open the axial gate and increase substrate accessibility into the degradation chamber. Most of this knowledge comes from the recent crystal structures of ClpP in complex with acyldepsipeptides (ADEP) antibiotics. These small molecules are providing new insights into the gating mechanism of this protease because they imitate the interaction of ClpA/ClpX with ClpP and activate its protease activity.

Mlh1-Mlh3, a Meiotic Crossover and DNA Mismatch Repair Factor, Is a Msh2-Msh3-stimulated Endonuclease

Background: Meiotic crossing over requires resolution of Holliday junctions through actions of the DNA mismatch repair factor Mlh1-Mlh3. Results: Mlh1-Mlh3 is a metal-dependent, Msh2-Msh3-stimulated endonuclease. Conclusion: Our observations support a direct role for Mlh1-Mlh3 endonuclease activity in recombination and repair. Significance: An enzymatic activity is identified for a key recombination and repair factor. Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair.

The PMS2 Subunit of Human MutLα Contains a Metal Ion Binding Domain of the Iron-Dependent Repressor Protein Family

DNA mismatch repair (MMR) is responsible for correcting replication errors. MutLalpha, one of the main players in MMR, has been recently shown to harbor an endonuclease/metal-binding activity, which is important for its function in vivo. This endonuclease activity has been confined to the C-terminal domain of the hPMS2 subunit of the MutLalpha heterodimer. In this work, we identify a striking sequence-structure similarity of hPMS2 to the metal-binding/dimerization domain of the iron-dependent repressor protein family and present a structural model of the metal-binding domain of MutLalpha. According to our model, this domain of MutLalpha comprises at least three highly conserved sequence motifs, which are also present in most MutL homologs from bacteria that do not rely on the endonuclease activity of MutH for strand discrimination. Furthermore, based on our structural model, we predict that MutLalpha is a zinc ion binding protein and confirm this prediction by way of biochemical analysis of zinc ion binding using the full-length and C-terminal domain of MutLalpha. Finally, we demonstrate that the conserved residues of the metal ion binding domain are crucial for MMR activity of MutLalpha in vitro.

The endonuclease domain of MutL interacts with the β sliding clamp

Mismatch repair corrects errors that have escaped polymerase proofreading enhancing replication fidelity by at least two orders of magnitude. The β and PCNA sliding clamps increase the polymerase processivity during DNA replication and are important at several stages of mismatch repair. Both MutS and MutL, the two proteins that initiate the mismatch repair response, interact with β. Binding of MutS to β is important to recruit MutS and MutL to foci. Moreover, the endonuclease activity of human and yeast MutLα is stimulated by PCNA. However, the concrete functions of the processivity clamp in the repair steps preceding DNA resynthesis remain obscure. Here, we demonstrate that the C-terminal domain of MutL encompasses a bona fide β-binding motif that mediates a weak, yet specific, interaction between the two proteins. Mutation of this conserved motif correlates with defects in mismatch repair, demonstrating that the direct interaction with β is important for MutL function. The interaction between the C-terminal domain of MutL and β is conserved in both Bacillus subtilis and Escherichia coli, but the repair defects associated with mutation of this β-binding motif are more severe in the former, suggesting that this interaction may have a more prominent role in methyl-independent than methyl-directed mismatch repair systems. Together with previously published data, our work strongly suggests that β may stimulate the endonuclease activity of MutL through its direct interaction with the C-terminal domain of MutL.

Crystal structure of a SeqA–N filament: implications for DNA replication and chromosome organization

Escherichia coli SeqA binds clusters of transiently hemimethylated GATC sequences and sequesters the origin of replication, oriC, from methylation and premature reinitiation. Besides oriC, SeqA binds and organizes newly synthesized DNA at replication forks. Binding to multiple GATC sites is crucial for the formation of stable SeqA–DNA complexes. Here we report the crystal structure of the oligomerization domain of SeqA (SeqA–N). The structural unit of SeqA–N is a dimer, which oligomerizes to form a filament. Mutations that disrupt filament formation lead to asynchronous DNA replication, but the resulting SeqA dimer can still bind two GATC sites separated from 5 to 34 base pairs. Truncation of the linker between the oligomerization and DNA‐binding domains restricts SeqA to bind two GATC sites separated by one or two full turns. We propose a model of a SeqA filament interacting with multiple GATC sites that accounts for both origin sequestration and chromosome organization.