Study of Interactions of DNA with RECA and Other Proteins.

This thesis describes different methods of stretching DNA using flow fields and investigates the aggregation behavior of RecA-DNA filaments in addition to the preliminary work on the kinetics of RecA-mediated strand exchanges. Using fluorescence microscopy, we compared DNA stretching by droplet evaporation, suctioning of a droplet with pipette tips, and blowing of a droplet with nitrogen or air and found that the best stretching was achieved in fast flows using suctioning and blowing. A statistical analysis of the average length and the surface deposition density of stretched lambda phage DNA molecules showed that both the hydrophobicity of the surface and the pH of the solution affect the stretching of DNA molecules greatly, but the hydrophobic interaction itself is not enough to explain the broad distribution of the stretch observed. The stretched DNA molecules can be used in the study of DNA/protein interactions, as exemplified by our observation of the motion of DNAse I during its interaction with stretched DNA molecules using a dual-color imaging system. We characterized the interwound or so-called “supercoiled” aggregates of RecA-DNA filaments using atomic force microscopy. Both RecA-dsDNA and RecA-ssDNA filaments showed mostly left-handedness and we found that single stranded DNA binding protein is necessary for the formation of ordered bundles of RecA-ssDNA filaments. We have suggested that the additional torsional stress generated when filaments approach each other is responsible for this observed coiling of RecA-DNA filaments. We reported the dual roles of ATP hydrolysis and RecA dissociation during strand exchange interactions based on the preliminary experimental observations of the two optimum conditions for fast strand exchanges. While the reaction with a length of 3.7kbp of incoming dsDNA yields maximum products with other conditions maintained, the reaction at 10mM phosphocreatine also produces most products compared with other concentrations. The observed optimums were correlated to the ATP hydrolysis and RecA dissociation during strand exchange interactions. The possible dual roles of these two steps could explain the observation of the two optimums.Ph.D.Chemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/58526/1/wshi_1.pd

BIOPHYSICAL STUDIES ON MECHANISMS OF HOMOLOGOUS RECOMBINATIONAL PROTEINS

Ph.DPH.D. IN MECHANOBIOLOGY (FOS

Recombinational Repair of a Chromosomal DNA Double Strand Break: A Dissertation

Repairing a chromosomal DNA double strand break is essential for survival and maintenance of genomic integrity of a eukaryotic organism. The eukaryotic cell has therefore evolved intricate mechanisms to counteract all sorts of genomic insults in the context of chromatin structure. Modulating chromatin structure has been crucial and integral in regulating a number of conserved repair processes along with other fundamental genomic processes like replication and transcription. The work in this dissertation has focused on understanding the role of chromatin remodeling enzymes in the repair of a chromosomal DNA double strand break by homologous recombination. This has been approached by recapitulating the biochemical formation of recombination intermediates on chromatin in vitro. In this study, we have demonstrated that the mere packaging of DNA into nucleosomal structure does not present a barrier for successful capture of homologous DNA sequences, a central step of the biochemical pathway of recombinational repair. It is only the assembly of heterochromatin-like more complex nucleo-protein structure that presents additional constraints to this key step. And, this additional constraint can be overcome by the activities of ATP-dependent chromatin remodeling enzymes. These findings have great implications for our perception of the mechanism of the recombinational repair process of a chromosomal DNA double strand break within the eukaryotic genome

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

Resolving stalled replication forks in Escherichia coli

DNA replication is essential to successful cell proliferation. Inheritance of traits during cell propagation relies on the accurate duplication of the parental double-stranded DNA (dsDNA) to form two identical daughter copies. This process is carried out by a multi-protein complex referred to as the replisome. Decades of investigations using the model Escherichia coli (E. coli) replisome have provided an overall picture of the process of DNA replication initiation, elongation and termination. However, DNA replication in cells occurs on template DNA coated in DNA-binding proteins that can act as roadblocks and stall the replisome, often resulting in drastic effects on the chromosome. However, the fate of the replisome at these sites remains poorly understood. Stalled DNA replication has been linked to the emergence of antimicrobial resistance in prokaryotes, and the development of severe physical disorders and diseases in eukaryotes. Therefore, understanding the underlying mechanisms of stalled DNA replication can inform future investigations into the maintenance of genome integrity. This thesis focuses on the development and use of single-molecule tools to investigate stalled replication and the resolution of protein roadblocks. Single-molecule tools provide the ability to watch one molecule at a time. Extensive use of these techniques has revealed the heterogeneity that exists within complex biological pathways. Specifically, this thesis highlights the myriad of previously unknown behaviors of proteins on DNA as revealed by single-molecule tools

DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein

[EN] Single-stranded DNA-binding proteins (SSBs) play a key role in genome maintenance, binding and organizing single-stranded DNA (ssDNA) intermediates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), present multiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentration. It has long been suggested that different binding modes might be used selectively for different functions. To study this possibility, we used optical tweezers to determine and compare the structure and energetics of long, individual HmtSSB¿DNA complexes assembled on preformed ssDNA and on ssDNA generated gradually during `in situ¿ DNA synthesis. We show that HmtSSB binds to preformed ss-DNA in two major modes, depending on salt and protein concentration. However, when protein binding was coupled to strand-displacement DNA synthesis, only one of the two binding modes was observed under all experimental conditions. Our results reveal a key role for the gradual generation of ssDNA in modulating the binding mode of a multimeric SSB protein and consequently, in generating the appropriate nucleoprotein structure for DNA synthetic reactions required for genome maintenance.We are grateful to Prof. M. Salas laboratory (CBMSO-CSIC) for generously providing the Phi29 DNA polymerase and to Juan P. García Villaluenga (UCM) for useful discussions. Spanish Ministry of Economy and Competitiveness [MAT2015-71806-R to J.R.A-G, FIS2010-17440, FIS2015-67765-R to F.J.C., BFU2012-31825, BFU2015-63714-R to B.I.]; Spanish Ministry of Education, Culture and Sport [FPU13/02934 to J.J., FPU13/02826 to E.B-H.]; National Institutes of Health [GM45925 to L.S.K.]; University of Tampere (to G.L.C.); Programa de Financiacion Universidad Complutense de Madrid-Santander Universidades [CT45/15-CT46/15 to F.C.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2015-63714-R].Morin, J.; Cerrón, F.; Jarillo, J.; Beltran-Heredia, E.; Ciesielski, G.; Arias-Gonzalez, JR.; Kaguni, L.... (2017). DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein. Nucleic Acids Research. 45(12):7237-7248. https://doi.org/10.1093/nar/gkx395S723772484512Shereda, R. D., Kozlov, A. G., Lohman, T. M., Cox, M. M., & Keck, J. L. (2008). 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Microsecond Dynamics of Protein–DNA Interactions: Direct Observation of the Wrapping/Unwrapping Kinetics of Single-stranded DNA around the E.coli SSB Tetramer. Journal of Molecular Biology, 359(1), 55-65. doi:10.1016/j.jmb.2006.02.070Lohman, T. M., & Ferrari, M. E. (1994). Escherichia Coli Single-Stranded DNA-Binding Protein: Multiple DNA-Binding Modes and Cooperativities. Annual Review of Biochemistry, 63(1), 527-570. doi:10.1146/annurev.bi.63.070194.002523Maier, D., Farr, C. L., Poeck, B., Alahari, A., Vogel, M., Fischer, S., … Schneuwly, S. (2001). Mitochondrial Single-stranded DNA-binding Protein Is Required for Mitochondrial DNA Replication and Development in Drosophila melanogaster. Molecular Biology of the Cell, 12(4), 821-830. doi:10.1091/mbc.12.4.821Ruhanen, H., Borrie, S., Szabadkai, G., Tyynismaa, H., Jones, A. W. E., Kang, D., … Yasukawa, T. (2010). 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Journal of Molecular Biology, 236(1), 165-178. doi:10.1006/jmbi.1994.1126Bhattacharyya, B., George, N. P., Thurmes, T. M., Zhou, R., Jani, N., Wessel, S. R., … Keck, J. L. (2013). Structural mechanisms of PriA-mediated DNA replication restart. Proceedings of the National Academy of Sciences, 111(4), 1373-1378. doi:10.1073/pnas.1318001111Carlini, L. E., Porter, R. D., Curth, U., & Urbanke, C. (1993). Viability and preliminary in vivo characterization of site directed mutants of Escherichia coli single-stranded DNA-binding protein. Molecular Microbiology, 10(5), 1067-1075. doi:10.1111/j.1365-2958.1993.tb00977.xGriffith, J. D., Harris, L. D., & Register, J. (1984). Visualization of SSB-ssDNA Complexes Active in the Assembly of Stable RecA-DNA Filaments. Cold Spring Harbor Symposia on Quantitative Biology, 49(0), 553-559. doi:10.1101/sqb.1984.049.01.062Morrical, S. W., & Cox, M. M. (1990). 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Binding configurations of single-stranded DNA binding protein and their influence on DNA recombinase

DNA inside a cell is continuously damaged through multiple mechanisms including environmental exposure to radiation, chemical agents, or UV light. Certain products of the cell's own metabolism, such as reactive oxygen species, can also damage the DNA. In the worst-case scenario, this damage results in double-stranded DNA (dsDNA) breaks. Double-stranded DNA breaks are lethal, and difficult to repair, with potential complications from genome rearrangement. To prevent this genetic instability, a cell can utilize a homologous chromosome as a template to accurately repair DSBs. This process is called homologous recombination. Homologous recombination begins when an enzyme complex binds to a blunt end of a dsDNA break. The complex unzips the dsDNA through its helicase activity, and simultaneously cleaves the newly-generated 5' end of the ssDNA. This process leaves the remaining ssDNA strand exposed to the surrounding environment and prone to nucleolytic and chemical attacks. Cells have evolved single-stranded DNA binding (SSB) proteins to wrap and protect this ssDNA. In E. coli, SSB is known to wrap ssDNA in a variety of binding configurations, or modes. Three different binding modes, (SSB)65, (SSB)56, and (SSB)35, which wraps 65, 56, and 35 nucleotides (nt) respectively, have been observed in vitro Previous studies have suggested that SSB binding in different modes may exhibit different levels of binding cooperativity. SSBs in the (SSB)65 binding mode form isolated clusters (limited cooperativity), while SSBs in the (SSB)35 binding mode form long filaments (unlimited cooperativity). These different levels of binding cooperativity have been proposed to be used selectively in different DNA metabolic processes, including DNA replication, recombination, and repair. In homologous recombination, recombinase RecA must bind and form nucleoprotein filaments on the ssDNA, in direct competition with SSB. Prior studies have shown that RecA is capable of forming filaments on ssDNA wrapped by SSBs in the (SSB)65 binding mode, but filament formation on ssDNA wrapped by SSBs in the (SSB)35 binding mode is inhibited. Recent single-molecule studies have been conducted to investigate this competitive process, but the detailed mechanisms remain unclear. Here, we use high-resolution optical tweezers with simultaneous fluorescence microscopy to observe directly the activity of ssDNA-SSB, ssDNA-RecA, and ssDNA-SSB-RecA complexes under tension, and characterize their mechanical properties. The instrument allows us to simultaneously probe and visualize the interactions of RecA and SSB with ssDNA in real time and with nanometer resolution. We confirm that individuals SSBs bind and compact ssDNA in discrete modes. Under low tension (1-3 pN), a single SSB wraps ssDNA in the (SSB)65 or (SSB)56 binding mode. At higher tension (4-8 pN), SSB exhibits transient wrapping-unwrapping, switching between the (SSB)56, (SSB)35, and (SSB)17 wrapping modes. When multiple SSBs are present on the ssDNA, the SSBs form isolated clusters in those solution conditions that favor the (SSB)65 binding mode. The configuration of the SSBs changes to a long and stable filament when solution conditions that favor the (SSB)35 binding mode are used. In the absence of SSB, RecAs nucleate filament rapidly on ssDNA. The nucleation rate of RecA is slowed down by several times when RecA is added to ssDNA coated with isolated clusters of SSBs in the (SSB)56 mode. The nucleation rate of RecA decreases further when long and stable filaments of SSBs in the (SSB)35 binding mode are present on the ssDNA. The same experiments also demonstrate that RecA is capable of removing these SSBs from the ssDNA in a step-wise manner. Our results reveal the importance of SSB binding modes and their oligomerization to DNA recombination, and further confirm that (SSB)65/(SSB)56 binding modes are more likely to facilitate the activity of recombinase RecA during the DNA repair. The (SSB)35 binding mode, on the contrary, inhibits RecA filament formation, and is believed to not play an important role in this recombination process

Choosing between DNA and RNA: the polymer specificity of RNA helicase NPH-II

NPH-II is a prototypical member of the DExH/D subgroup of superfamily II helicases. It exhibits robust RNA helicase activity, and a detailed kinetic framework for unwinding has been established. However, like most SF2 helicases, there is little known about its mode of substrate recognition and its ability to differentiate between RNA and DNA substrates. Here, we employ a series of chimeric RNA–DNA substrates to explore the molecular determinants for NPH-II specificity on RNA and to determine if there are conditions under which DNA is a substrate. We show that efficient RNA helicase activity depends exclusively on ribose moieties in the loading strand and in a specific section of the 3′-overhang. However, we also document the presence of trace activity on DNA polymers, showing that DNA can be unwound under extremely permissive conditions that favor electrostatic binding. Thus, while polymer-specific SF2 helicases control substrate recognition through specific interactions with the loading strand, alternative specificities can arise under appropriate reaction conditions

DNA Renaturation at the Water-Phenol Interface

We study DNA adsorption and renaturation in a water-phenol two-phase system, with or without shaking. In very dilute solutions, single-stranded DNA is adsorbed at the interface in a salt-dependent manner. At high salt concentrations the adsorption is irreversible. The adsorption of the single-stranded DNA is specific to phenol and relies on stacking and hydrogen bonding. We establish the interfacial nature of a DNA renaturation at a high salt concentration. In the absence of shaking, this reaction involves an efficient surface diffusion of the single-stranded DNA chains. In the presence of a vigorous shaking, the bimolecular rate of the reaction exceeds the Smoluchowski limit for a three-dimensional diffusion-controlled reaction. DNA renaturation in these conditions is known as the Phenol Emulsion Reassociation Technique or PERT. Our results establish the interfacial nature of PERT. A comparison of this interfacial reaction with other approaches shows that PERT is the most efficient technique and reveals similarities between PERT and the renaturation performed by single-stranded nucleic acid binding proteins. Our results lead to a better understanding of the partitioning of nucleic acids in two-phase systems, and should help design improved extraction procedures for damaged nucleic acids. We present arguments in favor of a role of phenol and water-phenol interface in prebiotic chemistry. The most efficient renaturation reactions (in the presence of condensing agents or with PERT) occur in heterogeneous systems. This reveals the limitations of homogeneous approaches to the biochemistry of nucleic acids. We propose a heterogeneous approach to overcome the limitations of the homogeneous viewpoint

The Role of Eukaryotic Recombinase Loop L1 During Homologous Recombination

Within the life of an organism, its deoxyribonucleic acid (DNA) is constantly bombarded with damaging agents from exogenous and endogenous sources. One of the most deleterious types of damage is the double-stranded break (DSB) in which a continuous strand of DNA is broken in two. As a result, the information stored in their connection is lost. If improperly repaired, a cell will either not survive or transform into a neoplasm. Homologous recombination (HR) is a mechanism by which the cell processes these broken ends and uses proteins called recombinases to search for an undamaged homologous DNA template for repairing the break, the homology search. Generally for eukaryotes, the recombinase, Rad51, performs the homology search. Without it, cells cannot repair spontaneous DSBs by recombination and instead, must use alternative, less efficacious pathways. This type of reparative homologous recombination generally occurs during mitosis and is thus called mitotic recombination. In addition to its role in repair, HR is employed by eukaryotes during the first stage of meiosis to create crossover events, or chiasmata, between DNA homologs. The formation of these chiasmata is necessary for proper segregation of the chromosomes, preventing aneuploidy in the haploid cells destined for sexual reproduction. These crossover events have an added evolutionary benefit of mixing genes between the parental chromosomes, creating allelic diversity in the haploid cells. Eukaryotes have evolved a subset of meioticallyexpressed proteins to mediate this process. Dmc1 is a meiosis-specific, second recombinase that eukaryotes require to properly form these crossover events between homologs. It is not entirely understood why most eukaryotes require a second recombinase specifically designed for meiotic HR. A potential reason for this second recombinase may lie in the preferred templates for recombination that Rad51 and Dmc1 seek. Rad51 is employed mitotically to repair spontaneous DSBs and thus searches for the perfect undamaged copy, the sister chromatid, to prevent the loss of genetic information. Conversely, Dmc1 is employ meiotically to purposely form crossover events between homologs, which carry single-nucleotide polymorphisms (SNPs) between parental chromosomes. Thus, Dmc1 must be able to anneal DNA strands that aren’t perfectly the same. This work uses the single-molecule technique of DNA curtains to understand the factors that effect Rad51 and Dmc1 homologous DNA-capture stability. The first part of Chapter 1 is a historical exploration of homologous recombination research and a review of the current understanding of the pathway. The second part of Chapter 1 discusses human diseases that are associated with the failure to properly repair double-strand breaks. Chapter 2 will explain the single-molecule DNA curtain technique used throughout this work. Chapter 3 will show that Dmc1 is more tolerant of mismatches in captured DNA than Rad51. Chapter 4 will test the limits of Dmc1’s tolerance to imperfect DNA and attempts understand how it accomplishes this tolerance. Chapter 5 will demonstrate that this tolerance of mismatches is mediated by a specific structural element in recombinases, loop L1, and a chimeric Rad51 with a Dmc1-like L1 can tolerate mismatches in vitro and in vivo. Chapter 6 will explore how recombinase mediators such as BARD1 and BRCA1 enhance RAD51’s ability to capture DNA during the homology search