RAD51 Protein ATP Cap Regulates Nucleoprotein Filament Stability

RAD51 mediates homologous recombination by forming an active DNA nucleoprotein filament (NPF). A conserved aspartate that forms a salt bridge with the ATP γ-phosphate is found at the nucleotide-binding interface between RAD51 subunits of the NPF known as the ATP cap. The salt bridge accounts for the nonphysiological cation(s) required to fully activate the RAD51 NPF. In contrast, RecA homologs and most RAD51 paralogs contain a conserved lysine at the analogous structural position. We demonstrate that substitution of human RAD51(Asp-316) with lysine (HsRAD51(D316K)) decreases NPF turnover and facilitates considerably improved recombinase functions. Structural analysis shows that archaebacterial Methanococcus voltae RadA(D302K) (MvRAD51(D302K)) and HsRAD51(D316K) form extended active NPFs without salt. These studies suggest that the HsRAD51(Asp-316) salt bridge may function as a conformational sensor that enhances turnover at the expense of recombinase activity

The Saccharomyces cerevisiae Srs2 Helicase Regulates Homologous Recombination through the Disassembly of Recombination Intermediates

Life on Earth relies on a set of instructions encoded within an organism’s genome that is passed along from one generation to the next. Inherent to this mechanism of propagation is the need to copy the genetic material before passing it along to the progeny. Errors in this process coupled with stochastic damage will inevitably lead to changes in these instructions and may result in a reduction of fitness or even death of an individual. Yet, these same changes are also responsible for the adaptation mandated by our dynamic environment. Thus, there exists a delicate balance between maintenance and alteration of genetic material that is embodied to a large part at the various intersections of DNA replication, recombination and repair. Homologous recombination (HR) has been well studied and found to play vital roles in many cellular processes from the repair of the harrowing double-stranded break, the restart of a stalled or collapsed replication fork, as well as proper chromosome segregation during meiosis, all with the goal of striking this delicate balance. And yet, while HR is incumbent for the fitness of an organism, if left unchecked this same process can become detrimental by preventing better suited DNA repair pathways, permanently arresting cell cycle progression and creating some of the very problems it was meant to address such as aneuploidy or cancer. Despite a wealth of knowledge, the precise regulatory mechanisms remain an active area of research as they provide likely targets to combat these persistent diseases. Motor proteins that translocate along DNA have been particularly compelling and elusive due to their transitory nature, as well as the inevitability of collisions with bound protein(s) or nucleic acid structures that are likely regulated intermediates in the process. The yeast Srs2 helicase/translocase has long been regarded as the prototypical “anti-recombinase” as it has been shown to dismantle the Rad51 presynaptic filament, but also displays contradictory pro-recombinase functions. In vivo studies of Srs2 have been hampered by its involvement in multiple bioprocesses beyond recombination, while bulk in vitro approaches often produce conflicting results. Recent single molecule imaging of these players has shed light onto their involvement in the regulation of the various stages of the canonical pathway of HR. The Greene laboratory has developed ssDNA curtains to study the pre-synaptic filament and shown that Rad51-ssDNA filaments can create bonafide D-loop intermediates that would be incapable of repair and thus represent a toxic intermediate. These structures persist far longer than the entire process of DSBR in vivo and led us to hypothesize that motor proteins would be a key regulatory element to dismantle improperly paired intermediates for redistribution of the bound proteins and reengagement of the homology search process. Here I extend the use of ssDNA curtains to study Srs2 as it assembles into multimeric complexes to perform long-range disruption of various pre- and post-synaptic filament assemblies that include replication protein A (RPA), Rad51, Rad52, and D-loops. For the first time, direct observation of Srs2 translocating over RPA filaments is provided and shows these proteins are efficiently removed by Srs2. By including Rad52 on the RPA filament, I offer a refined model of the contradictory pro- and anti-recombinase activities of Srs2 through its antagonism of the single-strand annealing pathway in favor of HR. Additionally, Srs2 was found to initiate Rad51 disruption at breaks in the continuity of the filament marked by the persistence of replication protein A (RPA), Rad52, or the presence of an improper D-loop intermediate, the latter of which is efficiently disrupted before continuing translocation. In contrast to the prevailing model, we demonstrate that direct interaction between Srs2 and Rad51 is not necessary for long-range Rad51 clearance. These findings offer insights into the dynamic regulation of crucial HR intermediates by Srs2 and demonstrate that sub-nuclear concentrations of these proteins may be a likely driver for their activities

Mechanics and execution of homologous recombination: a single-molecule view

Homologous recombination (HR) is an essential mechanism for the repair of toxic DNA double-strand breaks (DSBs), which, when not repaired accurately, can give rise to cancer and hereditary disorders. During HR, RAD51 forms helical nucleoprotein filaments on RPA-coated ssDNA with the help of mediator proteins (BRCA2 and RAD51 paralogs) and catalyses strand invasion into homologous duplex DNA. How this is achieved in not completely understood. To dissect the process on molecular level, I first reconstituted nematode RAD-51 presynaptic filament assembly in the presence of mediator proteins at the single-molecule level and demonstrated that BRC-2 promotes RAD-51 nucleation, while RAD-51 paralogs transiently bind 5’ RAD-51 filament ends to stimulate RAD-51 growth in a 3’ to 5’ direction. In the second part of the thesis, I investigated the consequences of a permanently ‘switching on’ RAD-51 by engineering a variant of human RAD51, I287T, that forms presynaptic complexes efficiently without the recombination mediators present and analysed its impact on cellular DNA metabolism. I showed that RAD51 I287T is toxic in cells as it interferes with genome duplication by promiscuously loading at replication forks. Lastly, I demonstrated that nematode RAD-51 is surprisingly tolerant to mismatches during DNA strand exchange catalysis. The mismatch tolerance can be abolished by engineering specific mutations into the DNA binding loop of RAD-51, which causes meiotic HR stalling in the absence of regulatory motor proteins. Together, this work has uncovered unappreciated mechanisms that promote and maintain optimal RAD51 filament assembly and how deviations to optimal assembly rates can lead to disease - a phenomenon referred to as the ‘Goldilocks principle’ of RAD51 assembly.Open Acces

Interrogation and engineering of RAD51:BRC repeat interactions

The maintenance of genetic information is a fundamental function of every organism. The DNA in every human cell endures thousands of lesions per day, and eukaryotes have developed a sophisticated response to tackle this damage. Double strand breaks (DSBs) are the most toxic type of DNA damage, potentially leading to chromosomal rearrangements and tumorigenesis. Homologous recombination (HR) is a repair pathway that uses a homologous template to faithfully repair the DSBs. Central to HR is a recombinase protein RAD51, which forms a nucleoprotein filament with the broken ends, and allows efficient homology search. RAD51 is a promising therapeutic target in oncology. BRCA2 is a crucial mediator of RAD51 function and interacts with RAD51 through a set of 8 BRC repeats. A BRC repeat consists of two modules, an FxxA and an LFDE module, each containing hot-spot residues that mediate binding. In this work, I set out to utilise these for the pharmacological targeting of human RAD51. First, using biophysical methods, I investigated a novel, high-affinity chimeric repeat BRC8-2 composed of FxxA and LFDE modules from different natural repeats. I determined the X-ray crystallographic structure of its complex, which allowed me to uncover the determinants of high affinity binding. I applied these findings to the design of novel macrocyclic peptide inhibitors of RAD51. A method for cysteine stapling of recombinantly produced peptides was optimised to yield the correct product of high purity. The optimised stapling methodology provides a promising general approach for recombinant production and evaluation of cysteine-stapled peptides. Peptides were rationally designed in a structure-guided manner, and a variety of stapling architectures were evaluated for binding. The resulting purified molecules exhibit high potency and are stable in serum. Further crystallographic studies shed light on how the stapling moiety affects the peptide binding mode. The derived peptides serve as novel modalities for targeting Rad51 protein-protein interactions and can inform subsequent development of therapeutic drugs. In addition to the human proteins, I investigated the BRC:RAD51 interactions in orthologs from Leishmania infantum, the causative agent of leishmaniasis. Using crystallography and biophysical methods, I uncovered novel features of BRC repeat binding. The presented work expands our understanding of the structural determinants of homologous recombination.MRC DT

Mechanism of regulation of human RAD51 recombinase through post translational modifications & mediator proteins

RAD51 protein plays an important role in homologous genetic recombination (HR), an essential DNA metabolic process used by cells to faithfully repair the most deleterious forms of DNA damage and maintain genomic integrity. RAD51 along with its bacterial counterpart RecA, bacteriophage UvsX and archaeal RadA have been subjected to genetic and biochemical scrutiny resulting in a plentitude of mechanistic and functional information on formation, regulation and activities of these recombinases. An important disconnect between these two lines of investigation still exists because the recombinase functions of RAD51 are highly regulated through mediator proteins like the BRCA2 recombination mediator, and a host of post translational modifications, namely phosphorylation. The mechanism and biochemical implications of these regulatory processes have not been satisfactorily evaluated in-vitro. This work characterizes the interaction between RAD51 and the BRCA2 recombination mediator protein using computational methods to generate homology models for this interaction which are validated through experimental data. Using the knowledge gained from our structural model for the RAD51 recombinase, I developed a novel strategy to understand several key mechanisms for the regulation of RAD51 by phosphorylation. RAD51 is phosphorylated by the cABL tyrosine kinase. The mechanistic and functional significance of this event is largely disputed. Using biochemical and single molecule assays reconstituting major activities of RAD51, I have successfully dissected the biochemical mechanism of regulation of RAD51 by the c-Abl kinase. The results of this work strongly correlate with observations made in previous cell based analysis

FUNCTIONAL INSIGHTS INTO RAD51 REGULATORY PROTEINS IN HOMOLOGOUS RECOMBINATION

Accurate repair of DNA is critical for genome stability and cancer prevention. DNA double-strand breaks are one of the most toxic lesions and can be repaired using the high-fidelity homologous recombination (HR) pathway. HR is highly conserved and uses a homologous template for repair. One central HR step is RAD51 nucleoprotein filament formation on the single-stranded DNA ends. RAD51 filament formation is required for the homology search and strand invasion steps of HR. RAD51 activity is tightly controlled by many positive and negative regulators, collectively termed the RAD51 mediators. The human RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and SWSAP1) are RAD51 mediator proteins that are highly conserved throughout eukaryotes and structurally resemble RAD51.They assemble into subcomplexes, BCDX2, CX3, and the Shu complex,to promote the HR activity of RAD51. The RAD51 mediators function to nucleate, elongate, stabilize, and disassemble RAD51 during repair. RAD51 paralog mutations are found in many breast and ovarian cancers as well as other cancer types. Despite their discovery three decades ago, few advances have been made in understanding their function. This work identified that the C. elegans Shu complex is functionally conserved from yeast and is composed of two RAD-51 paralogs, RFS-1 and RIP-1, which bind SWS-1. Disruption of the worm Shu complex results in DNA damage sensitivity and reduced RAD-51 foci showing for the first time that the Shu complex promotes HR in higher eukaryotes. In subsequent studies in human cell lines,this work demonstrated that disruption of the human RAD51 paralog, RAD51C,through cancer-associated point mutations can also cause DNA damage sensitivity and reduced HR. The conserved Walker A motif of human RAD51C is particularly important for maintaining protein-protein interactions of the BCDX2 and CX3 complexes and maintaining HR proficiency that we propose is critical for prevention of breast and ovarian cancers.Together, this work sheds light onto the function of the RAD51 regulators and their role in maintaining genome stability

The Shu complex is a conserved regulator of Rad51 filament formation

The budding yeast Shu complex, a heterotetramer of Shu1, Shu2, Csm2, and Psy3, is important for homologous recombination (HR)-mediated chromosome damage repair and was first characterized a decade ago as promoting Rad51-dependent HR in response to replicative stress, but its mechanistic function and conservation in eukaryotes has remained unknown. Here we provide evidence that the Shu complex is evolutionarily conserved throughout eukaryotes, where it is comprised of a clear Shu2 orthologue physically associating with Rad51 paralogues. The Shu complex itself physically interacts with the rest of the HR machinery during DNA damage repair. Finally, we uncover that the mechanistic function of the Shu complex as a stimulatory co-factor of Rad51 filament formation in vitro, likely explaining the in vivo function of the eukaryotic Shu complex in suppressing error-prone repair. Moving forward, our findings provide a framework for studying the function of the human Shu complex, which will have broad importance in our understanding of DNA damage repair

Design and characterization of LexA dimer interface mutants

Two key proteins, LexA and RecA, are involved in regulation of the SOS expression system in bacteria. LexA and RecA act as the transcriptional repressor and inducer of the SOS operon, respectively. LexA downregulates the expression of at least 43 unlinked genes and activated RecA interacts with the repressor LexA and therefore, LexA undergoes self-cleavage. The ability of the LexA protein to dimerize is critical for its ability to repress SOS-regulated genes in vivo, as the N-terminal domain (NTD) alone has a lower DNA-binding affinity without the C-terminal domain (CTD) and the components for the dimerization of LexA are located in the CTD. Two antiparallel β-strands (termed β-11) in the CTD at the dimer interface of LexA are involved in the dimerization. LexA interacts with the active form of RecA in vivo during the SOS response. It was determined experimentally that monomeric and non-cleavable LexA binds more tightly to RecA and is resistant to self-cleavage. Therefore, we reasoned that if we can produce such LexA mutants we would be able to stabilize the LexA and active RecA complex for crystallization. Therefore, in this experiment, we attempted to make a non-cleavable and predominantly monomeric LexA that interacts intimately with RecA. We produced four single mutations at the dimer interface of the non-cleavable and NTD-truncated mutant of LexA (∆68LexAK156A) in order to weaken the interactions at the interface. The predominant forms of LexA mutants and the affinities of interaction between the mutant LexA proteins and RecA were examined. ∆68LexAK156AR197P mutant was found as predominantly monomeric at a concentration of 33.3 μM both by gel filtration chromatography and dynamic light scattering (DLS) experiments. It also bound RecA more tightly than wild-type LexA. Another mutant, ∆68LexAK156AI196Y, was also found as predominantly monomeric at a concentration of 33.3 μM by DLS. Both these proteins were subjected to crystallization with wild-type RecA protein. We were able to produce some predominantly monomeric LexA with good binding affinity for RecA; however, we were unsuccessful in co-crystallization

ATP half-sites in RadA and RAD51 recombinases bind nucleotides.

Homologous recombination is essential for repair of DNA double-strand breaks. Central to this process is a family of recombinases, including archeal RadA and human RAD51, which form nucleoprotein filaments on damaged single-stranded DNA ends and facilitate their ATP-dependent repair. ATP binding and hydrolysis are dependent on the formation of a nucleoprotein filament comprising RadA/RAD51 and single-stranded DNA, with ATP bound between adjacent protomers. We demonstrate that truncated, monomeric Pyrococcus furiosus RadA and monomerised human RAD51 retain the ability to bind ATP and other nucleotides with high affinity. We present crystal structures of both apo and nucleotide-bound forms of monomeric RadA. These structures reveal that while phosphate groups are tightly bound, RadA presents a shallow, poorly defined binding surface for the nitrogenous bases of nucleotides. We suggest that RadA monomers would be constitutively bound to nucleotides in the cell and that the bound nucleotide might play a structural role in filament assembly.We would like to thank Dr Timothy Sharpe for help with MALS analysis of the monomeric RadA protein and Dr Tara Pukala for the mass spectrometric analysis of the same protein. We would like to thank X-ray crystallographic and Biophysics facilities at the Department of Biochemistry for access to their instrumentation. We thank Diamond Light Source for access to beamline I04 (proposal MX315), European Synchrotron Radiation Facility for access to beamline ID23-1 (proposal MX-705 17 and MX-857) and Swiss Light Source for access to beamline PXIII that contributed to the results presented here. This work was funded by Translational Award from the Wellcome Trust (080083/Z/06/Z).This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Wiley

RecA Regulation by RecU and DprA During Bacillus subtilis Natural Plasmid Transformation

Natural plasmid transformation plays an important role in the dissemination of antibiotic resistance genes in bacteria. During this process, Bacillus subtilis RecA physically interacts with RecU, RecX, and DprA. These three proteins are required for plasmid transformation, but RecA is not. In vitro, DprA recruits RecA onto SsbA-coated single-stranded (ss) DNA, whereas RecX inhibits RecA filament formation, leading to net filament disassembly. We show that a null recA (ΔrecA) mutation suppresses the plasmid transformation defect of competent ΔrecU cells, and that RecU is essential for both chromosomal and plasmid transformation in the ΔrecX context. RecU inhibits RecA filament growth and facilitates RecA disassembly from preformed filaments. Increasing SsbA concentrations additively contributes to RecU-mediated inhibition of RecA filament extension. DprA is necessary and sufficient to counteract the negative effect of both RecU and SsbA on RecA filament growth onto ssDNA. DprA-SsbA activates RecA to catalyze DNA strand exchange in the presence of RecU, but this effect was not observed if RecU was added prior to RecA. We propose that DprA contributes to RecA filament growth onto any internalized SsbA-coated ssDNA. When the ssDNA is homologous to the recipient, DprA antagonizes the inhibitory effect of RecU on RecA filament growth and helps RecA to catalyze chromosomal transformation. On the contrary, RecU promotes RecA filament disassembly from a heterologous (plasmid) ssDNA, overcoming an unsuccessful homology search and favoring plasmid transformation. The DprA–DprA interaction may promote strand annealing upon binding to the complementary plasmid strands and facilitating thereby plasmid transformation rather than through a mediation of RecA filament growth