No description available.

Homologous recombination is a fundamental cellular process evolved to maintain genomic integrity and to generate genetic diversity. It plays a crucial role in DNA repair, correct segregation of meiotic chromosomes and resumption of the stalled replication forks. In vitro, the homologous recombination pathway is kinetically separable into a four step process involving initiation, homologous pairing, branch migration and junction resolution. The process of pairing and strand exchange between two homologous double-stranded DNA molecules leads to the formation of an intermediate structure called the Holliday junction (HJ). The crucial enzyme involved in this step in bacteria is RecA. In eubacteria, the junction is processed by three proteins, collectively referred to as the RuvABC protein complex. RuvA binds to the HJ, while RuvB, a helicase, binds to the RuvA-HJ complex and pumps the duplex DNA thus facilitating branch migration. The work reported here is concerned with structural studies on mycobacterial RecA and RuvA. X-ray crystallography was used to solve the protein crystal structures. The hanging drop vapour diffusion method was used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator except for two data sets collected using synchrotron radiation. The data were processed mostly using Mosflm and Scala and few data sets were processed using the HKL program suite. The molecular replacement method using programs Phaser and AMoRe was used for structure solution. Structure refinements were carried out using programs CNS and PHENIX. Model building was performed using COOT and O. PROCHECK, MOLPROBITY, ALIGN and NACCESS were used for structure validation and analysis of the refined structures. Mycobacterium smegmatis RecA (MsRecA) and its nucleotide complexes crystallize in three different, but closely related, forms characterized by specific ranges of unit cell dimensions. The six crystals discussed in the earlier part of the thesis and the five reported earlier, all grown under the same or very similar conditions, belong to these three forms, all in space group P61. They include one obtained by reducing the relative humidity around the crystal. In all crystals, RecA monomers form filaments around a 61 screw axis. Thus, the c-dimension of the crystal corresponds to the pitch of the RecA filament. As reported in the case of E.coli RecA, the variation in the pitch among the three forms correlate well with the motion of the C-terminal domain of the RecA monomers with respect to the main domain. The domain motion is compatible with formation of inactive as well as active RecA filaments involving monomers with a fully ordered C-domain. It does not appear to influence the movement upon nucleotide-binding of the switch residue Gln 196, which is believed to provide the trigger for transmitting the effect of nucleotide-binding to the DNA-binding region. Interestingly, partial dehydration of the crystal results in the movement of the residue, in a way similar to that caused by nucleotide-binding. The ordering of the DNA-binding loops L1 and L2, which present an ensemble of conformations, is also unaffected by domain motion. The conformation of loop L2 appears to depend upon nucleotide-binding presumably on account of the movement of the switch residue which forms part of the loop. The conformations of loops L1 and L2 are correlated and have implications to intermolecular communications within the RecA filament. The structures resulting from different orientations of the C-domain and different conformations of the DNA-binding loops appear to represent snapshots of the RecA molecule at different phases of activity and provide insights into the mechanism of action of RecA. Crystal structures of mutants of MsRecA involving changes of Gln 196 from glutamine to alanine, asparagine and glutamic acid, wild type MsRecA and several of their nucleotide complexes were subsequently determined using mostly low temperature and partly room temperature X-ray data. At both the temperatures, nucleotide binding results in a movement of Gln 196 towards the bound nucleotide in the wild type protein. This movement is abolished in the mutants, thus establishing the structural basis for the triggering action of the residue in terms of the size, shape and the chemical nature of the side chain. The 25 crystal structures reported in this thesis, along with the 5 MsRecA structures reported earlier, provide further elaboration of the relation among the pitch of the `inactive´ RecA filament, the orientation of the C-terminal domain with respect to the main domain and the location of the switch residue. The low temperature structures define one extreme of the range of positions the C-domain can occupy. The movement of the C-domain is correlated to those of the LexA binding loop and the loop that connects the main and the N-terminal domains. These elements of molecular plasticity are made use of in the transition to the `active´ filament, as evidenced by the recently reported structures of RecA-DNA complexes. The available structures of RecA resulting from X-ray and electron microscopic studies appear to represent different stages in the trajectory of the allosteric transformations of the RecA filament. This work contributes to the description of the early stages of this trajectory and provides insights into structures relevant to the later stages. The interesting results observed in the case of MsRecA prompted similar studies on the RecA from Mycobacterium tuberculosis (MtRecA). In this study, the crystals were grown at slightly different conditions and examined at different relative humidities and temperatures. Surprisingly, in spite of the 92% sequence identity between the two proteins, the structures indicated MtRecA to be substantially less plastic than MsRecA. The crystal structures do not provide an obvious explanation for this difference. Further studies are warranted to explain the molecular basis of the difference. RuvA, along with RuvB, is involved in branch migration of heteroduplex DNA in homologous recombination. The structures of four crystal forms of RuvA from Mycobacterium tuberculosis (MtRuvA) have been determined. The RuvB-binding domain is cleaved off in one of them. Detailed models of the complexes of octameric RuvA from different species with the Holliday junction have also been constructed. A thorough examination of the structures determined as part of the doctoral programme and those reported earlier bring to light the hitherto unappreciated role of the RuvB-binding domain in determining inter-domain orientation and oligomerization. These structures also permit an exploration of the interspecies variability of structural features such as oligomerization and the conformation of the loop that carries the acidic pin, in terms of amino acid substitutions. These models emphasize the additional role of the RuvB-binding domain in HJ binding. This role along with its role in oligomerization could have important biological implications. In addition to the work on RecA and RuvA, which forms the body of the thesis, the author was also involved in a structural bioinformatics study in which several carbohydrate binding proteins were probed to identify common minimum principles required for binding mannose, glucose and galactose. The study, presented in an Appendix, identified interactions that were specific to particular sugars, leading to individual fingerprints. These fingerprints were then used for exploring lead compounds, using a fragment based approach. This investigation helped the author to familiarize himself with the analysis of protein structures and ligand design based on them.

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.

<i>Escherichia coli </i>RecA is a versatile protein that is involved in homologous recombination, and coordination of both the DNA damage response and translesion synthesis. Single-stranded DNA (ssDNA) that is generated at the site of double-stranded breaks serves as a signal to activate RecA. This allows RecA to form a long helical filament on the ssDNA, which is required in recombination, hydrolysis of ATP, and mediating the self-cleavage of some ser-lys dyad proteins such as the LexA repressor. In this thesis, the formation of the RecA/LexA complex did not require preactivation by ssDNA, instead a volume excluding agent in the presence of LexA was able to stimulate its formation. These preliminary results led to a hypothesis that the formation of the RecA/LexA complex is a thermodynamic process that involves three steps: (1) a change in RecAs conformation towards the active form, (2) a change in LexAs conformation towards the cleavable form (i.e. burial of the ser-lys dyad catalytic residues), and (3) the binding between the active form of RecA and the cleavable form of LexA. Evidence for this model was shown by the ability of either NaCl, LexA K156A, an ATP substrate, or a volume excluding agent to enhance the stability of the RecA/LexA complex, which was detected by both the ATPase and coprotease assays. Hyper-active RecA mutants, isolated form the yeast two-hybrid screen, were also tested, however they did not enhance the stability of the complex. Additionally, RecAs binding preference for the monomer or dimer form of LexA was examined, since it is unknown which species of LexA is able to enhance the stability of the complex. To generate the monomer form of LexA, single point mutations were introduced at the dimer interface of the protein such that its dimerization was disrupted by charge-charge repulsions. Based on the inhibition assay, RecA was found to bind preferentially to dimer form and not the monomer form of LexA, possible reasons for these results are discussed.

<i>Escherichia coli </i>RecA is a versatile protein that is involved in homologous recombination, and coordination of both the DNA damage response and translesion synthesis. Single-stranded DNA (ssDNA) that is generated at the site of double-stranded breaks serves as a signal to activate RecA. This allows RecA to form a long helical filament on the ssDNA, which is required in recombination, hydrolysis of ATP, and mediating the self-cleavage of some ser-lys dyad proteins such as the LexA repressor. In this thesis, the formation of the RecA/LexA complex did not require preactivation by ssDNA, instead a volume excluding agent in the presence of LexA was able to stimulate its formation. These preliminary results led to a hypothesis that the formation of the RecA/LexA complex is a thermodynamic process that involves three steps: (1) a change in RecAs conformation towards the active form, (2) a change in LexAs conformation towards the cleavable form (i.e. burial of the ser-lys dyad catalytic residues), and (3) the binding between the active form of RecA and the cleavable form of LexA. Evidence for this model was shown by the ability of either NaCl, LexA K156A, an ATP substrate, or a volume excluding agent to enhance the stability of the RecA/LexA complex, which was detected by both the ATPase and coprotease assays. Hyper-active RecA mutants, isolated form the yeast two-hybrid screen, were also tested, however they did not enhance the stability of the complex. Additionally, RecAs binding preference for the monomer or dimer form of LexA was examined, since it is unknown which species of LexA is able to enhance the stability of the complex. To generate the monomer form of LexA, single point mutations were introduced at the dimer interface of the protein such that its dimerization was disrupted by charge-charge repulsions. Based on the inhibition assay, RecA was found to bind preferentially to dimer form and not the monomer form of LexA, possible reasons for these results are discussed.

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.

No description available.

During the course of normal DNA replication, replication forks are constantly encountering "housekeeping" types of routine damage to the DNA template that may cause the forks to stall or collapse. One product of this fork collapse is the induction of the SOS response, a coordinated global response to help pause the growth and replication of a cell while DNA damage is addressed and repaired. In E. coli, this response is activated by the formation of ssDNA, to which the RecA protein binds and forms a nucleoprotein filament, which acts as the activator for autocleavage of the LexA transcriptional repressor, which normally represses expression of SOS genes. Damage responses are crucial to maintaining genomic integrity, and are therefore essential to all forms of life, and this type of regulatory system is highly conserved. However, cells have mechanisms for tightly regulating induction of these responses, and can often repair routine damage to their chromosomes without the need to induce SOS. This is chiefly evidenced by the observation that more than 20% of cells in a population have RecA filaments, but less than 1% are induced for SOS. How cells make this decision to induce SOS is the subject of this work. This dissertation describes three projects aimed at examining molecular mechanisms by which cells regulate RecA filaments, and therefore the decision to induce the SOS response. The first examines the disparity between the formation of RecA filaments, as evidenced by RecA-GFP foci, and the induction of SOS in the absence of damage, using a psulA-gfp reporter system. It is shown that there are three independent factors that repress SOS expression in undamaged E. coli cells. These are radA, the amount of recA in the cell, and in some circumstances recX. The first two limit SOS in wild type cells in the absence of external damage, while the third is an additional factor required in xthA mutants, likely due to the fact there are more RecA loading events in these mutants. These factors are thought to change the character and reduce the half-life and persistence of RecA filaments in the cell. The second project shows that suppression of SOS through the use of recA4162 and uvrD303 mutants is substrate and situation-specific. This specificity is demonstrated by the fact that, while both recA4162 and uvrD303 can suppress SOS in the SOS constitutive mutant recA730, recA4162 can only suppress SOS when the signal occurs at replication forks and not at any other place on the chromosome, while uvrD303 appears to suppress SOS with less specificity, and can suppress after UV (shown previously), at induced DSBs, and other places not directly at the replication fork. Here mutants of different replication factors are used that uncouple the replisome and induce SOS to a high degree. The third project determines the factors necessary for loading RecA filaments at the replication fork versus other locations on the chromosome when SOS is induced in the absence of damage, and helps elucidate further mechanisms for induction of SOS at these substrates. It is shown that the sbcB and recJ exonucleases assist in inappropriate RecA filament formation by substrate processing exclusively at replication forks, but not other substrates, likely through mechanisms that are reliant on the activities of the RecA loading factors RecBCD and RecFOR.

Maintaining genomic integrity is crucial for cell survival. In Escherichia coli, Rec-Amediated homologous recombination (HR) plays an essential role in the repair of DNA double-strand breaks (DSB) and the SOS response through a series of highly dynamic interactions with the chromosome. A greater understanding of the mechanism of homologous recombination requires quantitative analysis of genomic studies in live cells. The aim of this thesis was to investigate the dynamics of the RecA-DNA interactions in vivo following the induction of a site-specific DSB in the chromosome of E. coli. This DSB is caused by the cleavage of a DNA hairpin by the hairpin-specific endonuclease, SbcCD. The DNA hairpin is formed only on the lagging strand template of replication by a 246 bp-interrupted palindrome. As a result cleavage only occurs on one sister chromosome, leaving one unbroken chromosome to serve as a template for repair by HR. Here, this system has been used as a basis to develop a method that combines chromatin immunoprecipitation with quantitative PCR (ChIP-qPCR) and next-generation sequencing (ChIP-Seq) to quantify RecA protein binding during the active repair of a single chromosomal DSB. This study reports that DSB-dependent RecA binding is stimulated in response to the eight base DNA sequence Chi (5’-GCTGGTGG-3’). Increasing the number of Chi sites close to the DSB stimulates more RecA loading to DNA, with ChIP-Seq analysis also revealing a role for subsequent Chi sites in RecA binding during DSBR. If the Chi sites close to the DSB are removed then Chi-dependent RecA binding to DNA can be observed at distances greater than 100 kb from the DSB, suggesting that these subsequent Chi sites can be engaged in DSBR. Through collaboration, these in vivo data were combined with stochastic modeling to determine that, in vivo, Chi is recognised by the RecBCD complex with an efficiency of 20- 35%. The genomic analysis also revealed two unexpected aspects of RecA protein binding. First, ChIP-Seq analyses identified that following a DSB at lacZ there is RecA enrichment detected in the terminus region of the E. coli chromosome. This RecA binding is Chi-dependent, indicating a role for HR. Second, DSB-independent binding was observed at the RNA encoding genes dispersed throughout the chromosome. A temporal analysis of RecA dynamics was also performed. These analyses revealed that RecA binding to DNA near the DSB is extremely dynamic, cycling between periods of high RecA enrichment and periods of low RecA enrichment. This is the first in vivo study of DSB-dependent RecA-DNA distribution and dynamics in recombination proficient E. coli cells.

Made available in DSpace on 2017-07-21T19:25:54Z (GMT). No. of bitstreams: 1 Wellington Claiton Leite.pdf: 3789073 bytes, checksum: f4c16b4260fbd54f4eada652038ae5bc (MD5) Previous issue date: 2016-09-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The bacterial RecA protein plays a role in the complex system of DNA damage repair. In the presence of ATP, RecA proteins polymerize onto single-strand DNA (ssDNA) as righthanded helical nucleoprotein laments, and catalyze strand exchange reaction between the ssDNA and homologous double-strand DNA (dsDNA) molecules. These activities are supported or stimulated by accessory proteins, as the single-stranded binding protein (SSB).Here, we report a functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA).We report the crystal structure of HsRecA-ADP/ATP complex to 1.7 Å of atomic resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, similarly to homologous RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. The bacterial polymerization motif contains the sequence SV/IMR/KLG which interacts with the core ATPase domain residues DNLLLV/CS. In the inactive RecA, it is a loop - strand interaction, respectively, while in the active RecA it becomes a dyad strand. In both RecA forms, the polymerization motif seems to stabilize the subunitsubunit interface by hydrophobic interactions. The methionine of this motif may play an important role in the stability and formation of a right-handed helical nucleoprotein lament. The ATPase activity and the structure of the nucleoprotein lament of HsRecA and Escherichia coli RecA (EcRecA) were analyzed in the presence and absence of SSB. When SSB was added after RecA+ssDNA, HsRecA and EcRecA showed similar ATPase activity and nucleolament structure. However, when SSB was either not included or it was added before RecA+ssDNA, the HsRecA showed higher ATPase activity and formed longer nucleoprotein laments than EcRecA. Thus, HsRecA protein is more ecient at displacing SSB from ssDNA than EcRecA protein. HsRecA promoted DNA exchange more eficiently: a greater yield of nicked circular products were obtained in a shorter time. Reconstruction of electrostatic potential from the hexameric structure of HsRecAADP/ ATP revealed a high positive charge along the inner side, which is consistent with the fact that ssDNA binds inside the filament. It may explain the enhance capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote eficiently the DNA strand exchange reaction. Keywords: RecA, Crystallography, RecA nucleoprotein filament, ATPase activity, DNA strand exchange, crystal structure, structural analysis. / A proteína RecA bacteriana desempenha um importante papel no complexo sistema de reparo de danos ao DNA. Na presença de ATP, a proteína RecA se auto-polimeriza sobre o DNA simples ta (ssDNA) (do inglês single-strand DNA (ssDNA)) como um lamento de nucleoproteína helicoidal, cataliza a reação de troca de fitas entre as moléculas ssDNA e a ta de DNA dupla fita homóloga (dsDNA) (do inglês double-strand DNA (dsDNA)). Estas atividades são suportadas ou estimuladas por proteínas acessórias, como a proteína ligadora de ssDNA SSB (do inglês single-stranded binding protein (SSB)). Neste trabalho é apresentado a caracterização estrutural e funcional da proteína RecA da bactéria Herbaspirillum seropedicae. A estrutura tridimensional do complexo HsRecA-ADP/ATP foi resolvida numa resolução 1,7 Å. A estrutura monomérica da proteína HsRecA consiste em um pequeno domínio N-terminal, um domínio central contendo um sitío ATPásico e e um grande domínio C-terminal, similar com proteínas RecAs homólogas. Análises estruturais comparativas mostraram que o motivo de polimerização da região N-terminal de proteí- nas da familia RecA que incluem archaea e eucariotos, também está presente na proteína RecA bacteriana. O motivo de polimerização da região N-terminal de bactérias contêm a sequência de resíduos (Serina, Valina ou Isoleucina, Metionina, Arginina ou Lisina, Leucina, Glicina) que interage com a sequência de resíduos do core ATPásico (Aspartato, Asparagina, Leucina, Leucina, Leucina, Valina, Cisteína, Serina). Na proteína RecA inativa esta interação é do tipo loop - strand, respectivamente, enquanto na proteína RecA ativa essa interação se torna uma dupla -strand. Em ambas formas da RecA, o motivo de polimerização parece estabilizar a interface subunidade-subunidade por interações hidrofóbicas. No motivo N-terminal a presença de uma Metionina altamente conservada talvez desempenha um importante papel na estabilidade e formação do lamento de nucleoproteína. A atividade ATPásica e a estrutura do lamento de nucleoproteína da proteína HsRecA e da Escherichia coli RecA (EcRecA) foram analisadas na presença e ausência da proteína SSB. Quando a SSB foi adicionada após RecA+ssDNA, as proteínas HsRecA e EcRecA mostraram similar atividade ATPásica e estrutura de nucleo lamento. Entretanto, quando a SSB não estava incluída ou quando adicionada anteriormente a adição RecA+ssDNA, a proteína HsRecA mostrou maior atividade ATPásica e formou maiores lamentos de nucleoproteína que a proteína EcRecA. Ainda, a proteína HsRecA é mais eficiente em deslocar a SSB do ssDNA que a proteína EcRecA. A proteína HsRecA também promove a reação de troca de fitas mais eficientemente: uma maior quantidade de produtos duplex substrato convertido em duplex circular foram obtidos em um curto intervalo de tempo. A reconstrução do potencial eletrostático da estrutura hexamérica da proteína HsRecA revelou uma maior densidade de cargas positivas no seu interior, que é consistente com o fato que o ssDNA ligar-se internamente ao filamento hexamérico. Isto talvez possa explicar capacidade melhorada da proteína HsRecA ligar-se ao ssDNA, formando um continuo filamento de nucleoproteína, deslocando a SSB e ainda promovendo de forma eficiente a reação de trocas de fitas.