Local structural dynamics of Rad51 protomers revealed by cryo-electron microscopy of Rad51-ssDNA filaments.

Homologous recombination (HR) is a high-fidelity repair mechanism for double-strand breaks. Rad51 is the key enzyme that forms filaments on single-stranded DNA (ssDNA) to catalyze homology search and DNA strand exchange in recombinational DNA repair. In this study, we employed single-particle cryo-electron microscopy (cryo-EM) to ascertain the density map of the budding yeast Rad51-ssDNA filament bound to ADP-AlF 3 , achieving a resolution of 2.35 Å without imposing helical symmetry. The model assigned 6 Rad51 protomers, 24 nt of DNA, and 6 bound ADP-AlF 3 . It shows 6-fold symmetry implying monomeric building blocks, unlike the structure of the Rad51-I345T mutant filament with three-fold symmetry implying dimeric building blocks, for which the structural comparisons provide a satisfying mechanistic explanation. This image analysis enables comprehensive comparisons of individual Rad51 protomers within the filament and reveals local conformational movements of amino acid side chains. Notably, Arg293 in Loop1 adopts multiple conformations to facilitate Leu296 and Val331 in separating and twisting the DNA triplets. We also analyzed the predicted structures of yeast Rad51-K342E and two tumor-derived human RAD51 variants, RAD51-Q268P and RAD51-Q272L, using the Rad51-ssDNA structure from this study as a reference.

POLθ-mediated end joining is restricted by RAD52 and BRCA2 until the onset of mitosis.

BRCA2-mutant cells are defective in homologous recombination, making them vulnerable to the inactivation of other pathways for the repair of DNA double-strand breaks (DSBs). This concept can be clinically exploited but is currently limited due to insufficient knowledge about how DSBs are repaired in the absence of BRCA2. We show that DNA polymerase θ (POLθ)-mediated end joining (TMEJ) repairs DSBs arising during the S phase in BRCA2-deficient cells only after the onset of the ensuing mitosis. This process is regulated by RAD52, whose loss causes the premature usage of TMEJ and the formation of chromosomal fusions. Purified RAD52 and BRCA2 proteins both block the DNA polymerase function of POLθ, suggesting a mechanism explaining their synthetic lethal relationships. We propose that the delay of TMEJ until mitosis ensures the conversion of originally one-ended DSBs into two-ended DSBs. Mitotic chromatin condensation might further serve to juxtapose correct break ends and limit chromosomal fusions.

Cancer testis antigens and genomic instability: More than immunology.

Cancer testis antigens or genes (CTA, CTG) are predominantly expressed in adult testes while silenced in most or all somatic tissues with sporadic expression in many human cancers. Concerted misexpression of numerous CTA/CTGs is rarely observed. This finding argues against the germ cell theory of cancer. A surprising number of CTA/CTGs are involved in meiotic chromosome metabolism and specifically in meiotic recombination. Recent discoveries with a group of CTGs established that their misexpression in somatic cells results in genomic instability by interfering with homologous recombination (HR), a DNA repair pathway for complex DNA damage such as DNA double-stranded breaks, interstrand crosslinks, and single-stranded DNA gaps. HR-deficient tumors have specific vulnerabilities and show synthetic lethality with inhibition of polyADP-ribose polymerase, opening the possibility that expression of CTA/CTGs that result in an HR-defect could be used as an additional biomarker for HR status. Here, we review the repertoire of CTA/CTGs focusing on a cohort that functions in meiotic chromosome metabolism by interrogating relevant cancer databases and discussing recent discoveries.

Guardians of the Genome: BRCA2 and Its Partners.

The tumor suppressor BRCA2 functions as a central caretaker of genome stability, and individuals who carry BRCA2 mutations are predisposed to breast, ovarian, and other cancers. Recent research advanced our mechanistic understanding of BRCA2 and its various interaction partners in DNA repair, DNA replication support, and DNA double-strand break repair pathway choice. In this review, we discuss the biochemical and structural properties of BRCA2 and examine how these fundamental properties contribute to DNA repair and replication fork stabilization in living cells. We highlight selected BRCA2 binding partners and discuss their role in BRCA2-mediated homologous recombination and fork protection. Improved mechanistic understanding of how BRCA2 functions in genome stability maintenance can enable experimental evidence-based evaluation of pathogenic BRCA2 mutations and BRCA2 pseudo-revertants to support targeted therapy.

Turning end-joining upside down in mitosis.

How cells deal with DNA breaks during mitosis is not well understood. While canonical non-homologous end-joining predominates in interphase, it is inhibited in mitosis to avoid telomere fusions. DNA polymerase θ mediated end-joining appears to be repressed in interphase, but promotes break repair in mitosis. The nature and induction time of breaks might determine their fate during mitosis.

DSS1 and ssDNA regulate oligomerization of BRCA2.

The tumor suppressor BRCA2 plays a key role in initiating homologous recombination by facilitating RAD51 filament formation on single-stranded DNA. The small acidic protein DSS1 is a crucial partner to BRCA2 in this process. In vitro and in cells (1,2), BRCA2 associates into oligomeric complexes besides also existing as monomers. A dimeric structure was further characterized by electron microscopic analysis (3), but the functional significance of the different BRCA2 assemblies remains to be determined. Here, we used biochemistry and electron microscopic imaging to demonstrate that the multimerization of BRCA2 is counteracted by DSS1 and ssDNA. When validating the findings, we identified three self-interacting regions and two types of self-association, the N-to-C terminal and the N-to-N terminal interactions. The N-to-C terminal self-interaction of BRCA2 is sensitive to DSS1 and ssDNA. The N-to-N terminal self-interaction is modulated by ssDNA. Our results define a novel role of DSS1 to regulate BRCA2 in an RPA-independent fashion. Since DSS1 is required for BRCA2 function in recombination, we speculate that the monomeric and oligomeric forms of BRCA2 might be active for different cellular events in recombinational DNA repair and replication fork stabilization.

Saccharomyces cerevisiae Mus81-Mms4 prevents accelerated senescence in telomerase-deficient cells.

Alternative lengthening of telomeres (ALT) in human cells is a conserved process that is often activated in telomerase-deficient human cancers. This process exploits components of the recombination machinery to extend telomere ends, thus allowing for increased proliferative potential. Human MUS81 (Mus81 in Saccharomyces cerevisiae) is the catalytic subunit of structure-selective endonucleases involved in recombination and has been implicated in the ALT mechanism. However, it is unclear whether MUS81 activity at the telomere is specific to ALT cells or if it is required for more general aspects of telomere stability. In this study, we use S. cerevisiae to evaluate the contribution of the conserved Mus81-Mms4 endonuclease in telomerase-deficient yeast cells that maintain their telomeres by mechanisms akin to human ALT. Similar to human cells, we find that yeast Mus81 readily localizes to telomeres and its activity is important for viability after initial loss of telomerase. Interestingly, our analysis reveals that yeast Mus81 is not required for the survival of cells undergoing recombination-mediated telomere lengthening, i.e. for ALT itself. Rather we infer from genetic analysis that Mus81-Mms4 facilitates telomere replication during times of telomere instability. Furthermore, combining mus81 mutants with mutants of a yeast telomere replication factor, Rrm3, reveals that the two proteins function in parallel to promote normal growth during times of telomere stress. Combined with previous reports, our data can be interpreted in a consistent model in which both yeast and human MUS81-dependent nucleases participate in the recovery of stalled replication forks within telomeric DNA. Furthermore, this process becomes crucial under conditions of additional replication stress, such as telomere replication in telomerase-deficient cells.

Autism and Cancer Share Risk Genes, Pathways, and Drug Targets.

Autism is a neurodevelopmental disorder, diagnosed behaviorally by social and communication deficits, repetitive behaviors, and restricted interests. Recent genome-wide exome sequencing has revealed extensive overlap in risk genes for autism and for cancer. Understanding the genetic commonalities of autism(s) and cancer(s), with a focus on mechanistic pathways, could lead to repurposed therapeutics.

Regulation of recombination and genomic maintenance.

Recombination is a central process to stably maintain and transmit a genome through somatic cell divisions and to new generations. Hence, recombination needs to be coordinated with other events occurring on the DNA template, such as DNA replication, transcription, and the specialized chromosomal functions at centromeres and telomeres. Moreover, regulation with respect to the cell-cycle stage is required as much as spatiotemporal coordination within the nuclear volume. These regulatory mechanisms impinge on the DNA substrate through modifications of the chromatin and directly on recombination proteins through a myriad of posttranslational modifications (PTMs) and additional mechanisms. Although recombination is primarily appreciated to maintain genomic stability, the process also contributes to gross chromosomal arrangements and copy-number changes. Hence, the recombination process itself requires quality control to ensure high fidelity and avoid genomic instability. Evidently, recombination and its regulatory processes have significant impact on human disease, specifically cancer and, possibly, neurodegenerative diseases.

RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells.

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.

A conserved sequence extending motif III of the motor domain in the Snf2-family DNA translocase Rad54 is critical for ATPase activity.

Rad54 is a dsDNA-dependent ATPase that translocates on duplex DNA. Its ATPase function is essential for homologous recombination, a pathway critical for meiotic chromosome segregation, repair of complex DNA damage, and recovery of stalled or broken replication forks. In recombination, Rad54 cooperates with Rad51 protein and is required to dissociate Rad51 from heteroduplex DNA to allow access by DNA polymerases for recombination-associated DNA synthesis. Sequence analysis revealed that Rad54 contains a perfect match to the consensus PIP box sequence, a widely spread PCNA interaction motif. Indeed, Rad54 interacts directly with PCNA, but this interaction is not mediated by the Rad54 PIP box-like sequence. This sequence is located as an extension of motif III of the Rad54 motor domain and is essential for full Rad54 ATPase activity. Mutations in this motif render Rad54 non-functional in vivo and severely compromise its activities in vitro. Further analysis demonstrated that such mutations affect dsDNA binding, consistent with the location of this sequence motif on the surface of the cleft formed by two RecA-like domains, which likely forms the dsDNA binding site of Rad54. Our study identified a novel sequence motif critical for Rad54 function and showed that even perfect matches to the PIP box consensus may not necessarily identify PCNA interaction sites.

Synthetic lethality between gene defects affecting a single non-essential molecular pathway with reversible steps.

Systematic analysis of synthetic lethality (SL) constitutes a critical tool for systems biology to decipher molecular pathways. The most accepted mechanistic explanation of SL is that the two genes function in parallel, mutually compensatory pathways, known as between-pathway SL. However, recent genome-wide analyses in yeast identified a significant number of within-pathway negative genetic interactions. The molecular mechanisms leading to within-pathway SL are not fully understood. Here, we propose a novel mechanism leading to within-pathway SL involving two genes functioning in a single non-essential pathway. This type of SL termed within-reversible-pathway SL involves reversible pathway steps, catalyzed by different enzymes in the forward and backward directions, and kinetic trapping of a potentially toxic intermediate. Experimental data with recombinational DNA repair genes validate the concept. Mathematical modeling recapitulates the possibility of kinetic trapping and revealed the potential contributions of synthetic, dosage-lethal interactions in such a genetic system as well as the possibility of within-pathway positive masking interactions. Analysis of yeast gene interaction and pathway data suggests broad applicability of this novel concept. These observations extend the canonical interpretation of synthetic-lethal or synthetic-sick interactions with direct implications to reconstruct molecular pathways and improve therapeutic approaches to diseases such as cancer.

Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation.

Homologous recombination is a high-fidelity DNA repair pathway. Besides a critical role in accurate chromosome segregation during meiosis, recombination functions in DNA repair and in the recovery of stalled or broken replication forks to ensure genomic stability. In contrast, inappropriate recombination contributes to genomic instability, leading to loss of heterozygosity, chromosome rearrangements and cell death. The RecA/UvsX/RadA/Rad51 family of proteins catalyses the signature reactions of recombination, homology search and DNA strand invasion. Eukaryotes also possess Rad51 paralogues, whose exact role in recombination remains to be defined. Here we show that the Saccharomyces cerevisiae Rad51 paralogues, the Rad55-Rad57 heterodimer, counteract the antirecombination activity of the Srs2 helicase. The Rad55-Rad57 heterodimer associates with the Rad51-single-stranded DNA filament, rendering it more stable than a nucleoprotein filament containing Rad51 alone. The Rad51-Rad55-Rad57 co-filament resists disruption by the Srs2 antirecombinase by blocking Srs2 translocation, involving a direct protein interaction between Rad55-Rad57 and Srs2. Our results demonstrate an unexpected role of the Rad51 paralogues in stabilizing the Rad51 filament against a biologically important antagonist, the Srs2 antirecombination helicase. The biological significance of this mechanism is indicated by a complete suppression of the ionizing radiation sensitivity of rad55 or rad57 mutants by concomitant deletion of SRS2, as expected for biological antagonists. We propose that the Rad51 presynaptic filament is a meta-stable reversible intermediate, whose assembly and disassembly is governed by the balance between Rad55-Rad57 and Srs2, providing a key regulatory mechanism controlling the initiation of homologous recombination. These data provide a paradigm for the potential function of the human RAD51 paralogues, which are known to be involved in cancer predisposition and human disease.

Functions of the Snf2/Swi2 family Rad54 motor protein in homologous recombination.

Homologous recombination is a central pathway to maintain genomic stability and is involved in the repair of DNA damage and replication fork support, as well as accurate chromosome segregation during meiosis. Rad54 is a dsDNA-dependent ATPase of the Snf2/Swi2 family of SF2 helicases, although Rad54 lacks classical helicase activity and cannot carry out the strand displacement reactions typical for DNA helicases. Rad54 is a potent and processive motor protein that translocates on dsDNA, potentially executing several functions in recombinational DNA repair. Rad54 acts in concert with Rad51, the central protein of recombination that performs the key reactions of homology search and DNA strand invasion. Here, we will review the role of the Rad54 protein in homologous recombination with an emphasis on mechanistic studies with the yeast and human enzymes. We will discuss how these results relate to in vivo functions of Rad54 during homologous recombination in somatic cells and during meiosis. This article is part of a Special Issue entitled: Snf2/Swi2 ATPase structure and function.

Quality control of purified proteins involved in homologous recombination.

Biochemical reconstitution using purified proteins and defined DNA substrates is a key approach to develop a mechanistic understanding of homologous recombination. The introduction of sophisticated purification tags has greatly simplified the difficult task of purifying individual proteins or protein complexes, generating a wealth of mechanistic information. Using purified proteins in reconstituted recombination assays necessitates strict quality control to eliminate the possibility that relevant protein or nucleic acid contaminations lead to misinterpretation of experimental data. Here we provide simple protocols that describe how to detect in purified protein preparations contaminating nucleic acids and relevant enzymatic activities that may interfere with in vitro recombination assays. These activities include ATPases, indicating the potential presence of helicases or translocases, endo- and exonucleases, phosphatases, and type I or type II topoisomerases.

Presynaptic filament dynamics in homologous recombination and DNA repair.

Homologous recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments-helical filaments of a recombinase enzyme bound to single-stranded DNA (ssDNA). Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we reviewed the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments; some intrinsic such as recombinase ATP-binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examined dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examined the biochemical properties of recombination proteins from four model systems (T4 phage, Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We proposed that the presynaptic filament has evolved to rely on multiple external factors for increased multilevel regulation of HR processes in genomes with greater structural and sequence complexity.

Human BRCA2 protein promotes RAD51 filament formation on RPA-covered single-stranded DNA.

BRCA2 is a tumor suppressor that functions in homologous recombination, a key genomic integrity pathway. BRCA2 interacts with RAD51, the central protein of recombination, which forms filaments on single-stranded DNA (ssDNA) to perform homology search and DNA strand invasion. We report the purification of full-length human BRCA2 and show that it binds to ~6 RAD51 molecules and promotes RAD51 binding to ssDNA coated by replication protein A (RPA), in a manner that is stimulated by DSS1.

Regulation of homologous recombination in eukaryotes.

Homologous recombination (HR) is required for accurate chromosome segregation during the first meiotic division and constitutes a key repair and tolerance pathway for complex DNA damage, including DNA double-strand breaks, interstrand crosslinks, and DNA gaps. In addition, recombination and replication are inextricably linked, as recombination recovers stalled and broken replication forks, enabling the evolution of larger genomes/replicons. Defects in recombination lead to genomic instability and elevated cancer predisposition, demonstrating a clear cellular need for recombination. However, recombination can also lead to genome rearrangements. Unrestrained recombination causes undesired endpoints (translocation, deletion, inversion) and the accumulation of toxic recombination intermediates. Evidently, HR must be carefully regulated to match specific cellular needs. Here, we review the factors and mechanistic stages of recombination that are subject to regulation and suggest that recombination achieves flexibility and robustness by proceeding through metastable, reversible intermediates.