LUCA PELLEGRINI



Macromolecular assemblies and mechanisms of DNA replication and repair




A Ctf4 trimer couples the CMG helicase to DNA polymerase alpha in the eukaryotic replisome
Simon AC, Zhou JC, Perera RL, van Deursen F, Evrin C, Ivanova ME, Kilkenny ML, Renault L, Kjaer S, Matak-Vinković D, Labib K, Costa A, Pellegrini L. Nature 2014 May 4. doi: 10.1038/nature13234. [Epub ahead of print]
[PubMed][PDF]
Coordinates: [4C8H][4C8S][4C93][4c95]
Ctf4 trimer
Efficient duplication of the genome requires the concerted action of helicase and DNA polymerases at replication forks, to avoid stalling of the replication machinery and consequent genomic instability. In eukaryotes, the physical coupling between helicase and DNA polymerases remains poorly understood. Here we define the molecular mechanism by which the yeast Ctf4 protein links the Cdc45-MCM-GINS (CMG) DNA helicase to DNA polymerase alpha (Pol alpha) within the replisome. We use crystallography and electron microscopy to show that Ctf4 self-associates in a constitutive disk-shaped trimer. Trimerization depends on a beta-propeller domain in the carboxy-terminal half of the protein, which is fused to a helical extension that protrudes from one face of the trimeric disk. Critically, Pol alpha and the CMG helicase share a common mechanism of interaction with Ctf4. We show that the N-terminal tails of the catalytic subunit of Pol alpha and the Sld5 subunit of GINS contain a conserved Ctf4-binding motif that docks onto the exposed helical extension of a Ctf4 protomer within the trimer. Accordingly, we demonstrate that one Ctf4 trimer can support binding of up to three partner proteins, including the simultaneous association with both Pol alpha and GINS. Our findings indicate that Ctf4 can couple two molecules of Pol alpha to one CMG helicase within the replisome, providing a new paradigm for lagging-strand synthesis in eukaryotes that resembles the emerging model for the simpler replisome of E. coli. The ability of Ctf4 to act as a platform for multivalent interactions illustrates a mechanism for the concurrent recruitment of factors that act together at the fork.


Structures of human primase reveal design of nucleotide elongation site and mode of Pol α tethering.
Kilkenny ML, Longo MA, Perera RL, Pellegrini L. PNAS 2013 110 15961-6
[PubMed][PDF]
Coordinates: [4BPU][4BPX][4BPW]
Primase
Initiation of DNA synthesis in genomic duplication depends on primase, the DNA-dependent RNA polymerase that synthesizes de novo the oligonucleotides that prime DNA replication. Due to the discontinuous nature of DNA replication, primase activity on the lagging strand is required throughout the replication process. In eukaryotic cells, the presence of primase at the replication fork is secured by its physical association with DNA polymerase α (Pol α), which extends the RNA primer with deoxynucleotides. Our knowledge of the mechanism that primes DNA synthesis is very limited, as structural information for the eukaryotic enzyme has proved difficult to obtain. Here, we describe the crystal structure of human primase in heterodimeric form consisting of full-length catalytic subunit and a C-terminally truncated large subunit. We exploit the crystallographic model to define the architecture of its nucleotide elongation site and to show that the small subunit integrates primer initiation and elongation within the same set of functional residues. Furthermore, we define in atomic detail the mode of association of primase to Pol α, the critical interaction that keeps primase tethered to the eukaryotic replisome.


Mechanism for priming DNA synthesis by yeast DNA polymerase α.
Perera RL, Torella R, Klinge S, Kilkenny ML, Maman JD, Pellegrini L. eLife 2013 2 00482
[PubMed][PDF]
Coordinates: [4B08][4FVM][4FXD][4FYD]
Pol alpha
The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment.


Structural analysis of the human SYCE2-TEX12 complex provides molecular insights into synaptonemal complex assembly.
Davies OR, Maman JD, Pellegrini L. Open Biol 2012 2 120099
[PubMed][PDF]
The human SYC2-TEX12 complex
The successful completion of meiosis is essential for all sexually reproducing organisms. The synaptonemal complex (SC) is a large proteinaceous structure that holds together homologous chromosomes during meiosis, providing the structural framework for meiotic recombination and crossover formation. Errors in SC formation are associated with infertility, recurrent miscarriage and aneuploidy. The current lack of molecular information about the dynamic process of SC assembly severely restricts our understanding of its function in meiosis. Here, we provide the first biochemical and structural analysis of an SC protein component and propose a structural basis for its function in SC assembly. We show that human SC proteins SYCE2 and TEX12 form a highly stable, constitutive complex, and define the regions responsible for their homotypic and heterotypic interactions. Biophysical analysis reveals that the SYCE2-TEX12 complex is an equimolar hetero-octamer, formed from the association of an SYCE2 tetramer and two TEX12 dimers. Electron microscopy shows that biochemically reconstituted SYCE2-TEX12 complexes assemble spontaneously into filamentous structures that resemble the known physical features of the SC central element (CE). Our findings can be combined with existing biological data in a model of chromosome synapsis driven by growth of SYCE2-TEX12 higher-order structures within the CE of the SC.


A conserved motif in the C-terminal tail of DNA polymerase α tethers primase to the eukaryotic replisome.
Kilkenny ML, De Piccoli G, Perera RL, Labib K, Pellegrini L. J Biol Chem. 2012 287 23740-7
[PubMed] [PDF]
Pol alpha C-tail.png
The DNA polymerase α/primase complex forms an essential part of the eukaryotic replisome. The catalytic subunits of primase and Pol α synthesise composite RNA-DNA primers that initiate the leading and lagging DNA strands at replication forks. The physical basis and physiological significance of tethering primase to the eukaryotic replisome via Pol α remain poorly characterised. We have identified a short conserved motif at the extreme C-terminus of Pol α that is critical for interaction of the yeast orthologue Pol1 with primase. We show that truncation of the C-terminal residues 1452-1468 of Pol1 abrogates the interaction with the primase, as does mutation to alanine of the invariant amino acid F1463. Conversely, a Pol1 peptide spanning the last 16 residues binds primase with high affinity, and the equivalent peptide from human Pol α binds primase in an analogous fashion. These in vitro data are mirrored by experiments in yeast cells, as primase does not interact in cell extracts with Pol1 that either terminates at residue 1452 or has the F1463A mutation. The ability to disrupt the association between primase and Pol α allowed us to assess the physiological significance of primase being tethered to the eukaryotic replisome in this way. We find that the F1463A mutation in Pol1 renders yeast cells dependent on the S-phase checkpoint, whereas truncation of Pol1 at amino acid 1452 blocks yeast cell proliferation. These findings indicate that tethering of primase to the replisome by Pol α is critical for the normal action of DNA replication forks in eukaryotic cells.


Structural and functional insights into DNA-end processing by the archaeal HerA helicase-NurA nuclease complex.
Blackwood JK, Rzechorzek NJ, Abrams AS, Maman JD, Pellegrini L, Robinson NP. Nucleic Acids Res. 2012 40 3183-96
[PubMed] [PDF][Coordinates]

NurA.jpg

Helicase-nuclease systems dedicated to DNA end resection in preparation for homologous recombination (HR) are present in all kingdoms of life. In thermophilic archaea, the HerA helicase and NurA nuclease cooperate with the highly conserved Mre11 and Rad50 proteins during HR-dependent DNA repair. Here we show that HerA and NurA must interact in a complex with specific subunit stoichiometry to process DNA ends efficiently. We determine crystallographically that NurA folds in a toroidal dimer of intertwined RNaseH-like domains. The central channel of the NurA dimer is too narrow for double-stranded DNA but appears well suited to accommodate one or two strands of an unwound duplex. We map a critical interface of the complex to an exposed hydrophobic epitope of NurA abutting the active site. Based upon the presented evidence, we propose alternative mechanisms of DNA end processing by the HerA-NurA complex.


Flexible tethering of primase and DNA Pol α in the eukaryotic primosome.
Núñez-Ramírez R, Klinge S, Sauguet L, Melero R, Recuero-Checa MA, Kilkenny M, Perera RL, García-Alvarez B, Hall RJ, Nogales E, Pellegrini L, Llorca O. Nucleic Acids Res. 2011, 39, 8187-99. [PubMed] [PDF]

NurA.jpg

The Pol α/primase complex or primosome is the primase/polymerase complex that initiates nucleic acid synthesis during eukaryotic replication. Within the primosome, the primase synthesizes short RNA primers that undergo limited extension by Pol α. The resulting RNA-DNA primers are utilized by Pol δ and Pol ε for processive elongation on the lagging and leading strands, respectively. Despite its importance, the mechanism of RNA-DNA primer synthesis remains poorly understood. Here, we describe a structural model of the yeast primosome based on electron microscopy and functional studies. The 3D architecture of the primosome reveals an asymmetric, dumbbell-shaped particle. The catalytic centers of primase and Pol α reside in separate lobes of high relative mobility. The flexible tethering of the primosome lobes increases the efficiency of primer transfer between primase and Pol α. The physical organization of the primosome suggests that a concerted mechanism of primer hand-off between primase and Pol α would involve coordinated movements of the primosome lobes. The first three-dimensional map of the eukaryotic primosome at 25 Å resolution provides an essential structural template for understanding initiation of eukaryotic replication.


Shared active site architecture between the large subunit of eukaryotic primase and DNA photolyase.
Sauguet L, Klinge S, Perera RL, Maman JD, Pellegrini L. PLoS One 2010, 5:e10083. [PubMed] [PDF][Coordinates]

DNA synthesis during replication relies on RNA primers synthesised by the primase, a specialised DNA-dependent RNA polymerase that can initiate nucleic acid synthesis de novo. In archaeal and eukaryotic organisms, the primase is a heterodimeric enzyme resulting from the constitutive association of a small (PriS) and large (PriL) subunit. The ability of the primase to initiate synthesis of an RNA primer depends on a conserved Fe-S domain at the C-terminus of PriL (PriL-CTD). However, the critical role of the PriL-CTD in the catalytic mechanism of initiation is not understood. Here we report the crystal structure of the yeast PriL-CTD at 1.55 A resolution. The structure reveals that the PriL-CTD folds in two largely independent alpha-helical domains joined at their interface by a [4Fe-4S] cluster. The larger N-terminal domain represents the most conserved portion of the PriL-CTD, whereas the smaller C-terminal domain is largely absent in archaeal PriL. Unexpectedly, the N-terminal domain reveals a striking structural similarity with the active site region of the DNA photolyase/cryptochrome family of flavoproteins. The region of similarity includes PriL-CTD residues that are known to be essential for initiation of RNA primer synthesis by the primase. Our study reports the first crystallographic model of the conserved Fe-S domain of the archaeal/eukaryotic primase. The structural comparison with a cryptochrome protein bound to flavin adenine dinucleotide and single-stranded DNA provides important insight into the mechanism of RNA primer synthesis by the primase.


alpha CTD - B subunit complex.jpg

3D architecture of DNA Pol alpha reveals the functional core of multi-subunit replicative polymerases.
Klinge S, Núñez-Ramírez R, Llorca O, Pellegrini L. EMBO J. 2009 28 1978-87 [PubMed][PDF][Coordinates]

Eukaryotic DNA replication requires the coordinated activity of the multi-subunit DNA polymerases: Pol alpha, Pol delta and Pol epsilon. The conserved catalytic and regulatory B subunits associate in a constitutive heterodimer that represents the functional core of all three replicative polymerases. Here, we combine X-ray crystallography and electron microscopy (EM) to describe subunit interaction and 3D architecture of heterodimeric yeast Pol alpha. The crystal structure of the C-terminal domain (CTD) of the catalytic subunit bound to the B subunit illustrates a conserved mechanism of accessory factor recruitment by replicative polymerases. The EM reconstructions of Pol alpha reveal a bilobal shape with separate catalytic and regulatory modules. Docking of the B-CTD complex in the EM reconstruction shows that the B subunit is tethered to the polymerase domain through a structured but flexible linker. Our combined findings provide a structural template for the common functional architecture of the three major replicative DNA polymerases.


Structural basis for inhibition of homologous recombination by the RecX protein.
Ragone S, Maman JD, Furnham N, Pellegrini L. EMBO J. 2008 27 2259-69 [PubMed][PDF][Coordinates]

RecX.jpeg

The RecA/RAD51 nucleoprotein filament is central to the reaction of homologous recombination (HR). Filament activity must be tightly regulated in vivo as unrestrained HR can cause genomic instability. Our mechanistic understanding of HR is restricted by lack of structural information about the regulatory proteins that control filament activity. Here, we describe a structural and functional analysis of the HR inhibitor protein RecX and its mode of interaction with the RecA filament. RecX is a modular protein assembled of repeated three-helix motifs. The relative arrangement of the repeats generates an elongated and curved shape that is well suited for binding within the helical groove of the RecA filament. Structure-based mutagenesis confirms that conserved basic residues on the concave side of RecX are important for repression of RecA activity. Analysis of RecA filament dynamics in the presence of RecX shows that RecX actively promotes filament disassembly. Collectively, our data support a model in which RecX binding to the helical groove of the filament causes local dissociation of RecA protomers, leading to filament destabilisation and HR inhibition.



EPR spectrum of PriL-CTD.jpg

An iron-sulfur domain of the eukaryotic primase is essential for RNA primer synthesis.
Klinge S, Hirst J, Maman JD, Krude T, Pellegrini L. Nat Struct Mol Biol. 2007, 14, 875-7 [PubMed][PDF]

Primases synthesize the RNA primers that are necessary for replication of the parental DNA strands. Here we report that the heterodimeric archaeal/eukaryotic primase is an iron-sulfur (Fe-S) protein. Binding of the Fe-S cluster is mediated by an evolutionarily conserved domain at the C terminus of the large subunit. We further show that the Fe-S domain is essential to the unique ability of the eukaryotic primase to start DNA replication.






Interaction with the BRCA2 C terminus protects RAD51-DNA filaments from disassembly by BRC repeats.
Davies OR, Pellegrini L. Nat Struct Mol Biol. 2007, 14, 475-83 [PubMed][PDF]

BRCA2 has an essential function in DNA repair by homologous recombination, interacting with RAD51 via short motifs in the middle and at the C terminus of BRCA2. Here, we report that a conserved 36-residue sequence of human BRCA2 encoded by exon 27 (BRCA2Exon27) interacts with RAD51 through the specific recognition of oligomerized RAD51 ATPase domains. BRCA2Exon27 binding stabilizes the RAD51 nucleoprotein filament against disassembly by BRC repeat 4. The protection is specific for RAD51 filaments formed on single-stranded DNA and is lost when BRCA2Exon27 is phosphorylated on Ser3291. We propose that productive recombination results from the functional balance between the different RAD51-binding modes [corrected] of the BRC repeat and exon 27 regions of BRCA2. Our results further suggest a mechanism in which CDK phosphorylation of BRCA2Exon27 at the G2-M transition alters the balance in favor of RAD51 filament disassembly, thus terminating recombination.

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PAST RESEARCH

Structural insights into homologous recombination from the structure of a RAD51 - BRCA2 complexRAD51 core - BRCA2 BRC4 complex [PDF]
[medline] [coordinates (PDBid: 1N0W)]

The breast cancer susceptibility protein BRCA2 controls the activity of the RAD51 recombinase in pathways that lead to DNA repair by homologous recombination. The structure of the RAD51 RecA-homology domain bound to BRC repeat 4 of BRCA2 shows that the BRC repeat mimicks a short RAD51 motif used by the recombinase to self-associate into a nucleoprotein filament, an essential intermediate of genetic recombination. BRCA2 thus controls RAD51 activity by acting as a binding antagonist to RAD51 filament formation.

Comments in BBC news, Nature Structural Biology and Nature Reviews Cancer




Structural basis for the interaction of Xrcc4 with DNA Ligase IVXrcc4 core bound to DNA ligase IV linker region [PDF] [medline] [coordinates (PDBid: 1IK9)]

The Xrcc4 and DNA Ligase IV proteins are essential for non-homologous joining of DNA ends, an important mechanism of DNA double strand break repair. They are usually found associated in a tight complex in the cells. We have solved the structure of the proteolytically resistant core of Xrcc4 bound to its recognition site on DNA Ligase IV. The amino acid conservation in the two proteins is strongest at the complex interface, providing evidence that the observed mode of interaction has been maintained by the non-homologous end joining apparatus throughout evolution.