Single-stranded DNA binding proteins (SSB) bind to single-stranded DNA (ssDNA) that is generated by molecular machines such as helicases and polymerases. SSBs play crucial roles in DNA translation, replication and repair and their importance is demonstrated by their inclusion across all domains of life. The homotetrameric E. coli SSB and the heterotrimeric human RPA demonstrate how SSBs can vary structurally, but all fulfil their roles by employing oligonucleotide/oligosaccharide binding (OB) folds. Nucleofilaments of SSB proteins bound to ssDNA sequester the ssDNA strands, and in doing so protect exposed bases, keep the ssDNA in conformations favoured by other proteins that metabolise DNA and also recruit other proteins to bind to ssDNA. This thesis focuses on the SSB from the archaeon S. solfataricus (SsoSSB), and has found SsoSSB to be a monomer that binds cooperatively to ssDNA with a binding site size of 4-5 nucleotides. Tagging ssDNA and SsoSSB with fluorescent labels allowed the real time observation of single molecule interactions during the initial nucleation event and subsequent binding of an adjacent SsoSSB monomer. This was achieved by interpreting fluorescent traces that have recorded combinations of FRET, protein induced fluorescent enhancement (PIFE) and quenching events. This novel analysis gave precise measurements of the dynamics of the first and second monomers binding to ssDNA, which allowed affinity and cooperativity constants to be quantified for this important molecular process. SsoSSB was also found to have a similar affinity for RNA, demonstrating a promiscuity not found in other SSBs and suggesting further roles for SsoSSB in the cell - possibly exploiting its capacity to protect nucleic acids from degradation. The extreme temperatures that S. solfataricus experiences and the strength of the interaction with ssDNA and RNA make exploring the application of SsoSSB for industrial uses an interesting prospect; and its rare monomeric structure provides an opportunity to investigate the action of OB folds in a more isolated environment than in higher order structures.

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To maintain the genomic integrity, cell has evolved various DNA repair pathways. Base Excision Repair pathway (BER) is one such DNA repair pathway which is dedicated to protect DNA from small lesions such as oxidation, alkylation, deamination and loss of bases. Uracil is a promutagenic base which appears in the genome as a result of misincorporation of dUTP or due to oxidative deamination of cytosine. Uracil-DNA glycosylases (UDGs) are DNA repair enzymes that initiate multistep base excision repair (BER) pathway to excise uracil from DNA. Excision of uracil generates an abasic site (APDNA). AP-sites are cytotoxic and mutagenic to the cell. AP endonucleases act downstream to UDG in this pathway and generate substrates for DNA polymerase to fill in the correct bases. The cytotoxicity of AP-sites raises the question whether uracil excision activity is coupled to AP endonuclease activity. Also, there is transient formation of single stranded DNA (ssDNA) during DNA metabolic processes such as replication, repair and recombination. ssDNA is more prone to various nucleases and DNA damaging agents. All the living organisms encode single stranded DNA binding protein (SSB) that binds to ssDNA and protects it from various damages. In addition, SSB plays a vital role during DNA replication, repair and recombination. Studies on SSBs from prototype Escherichia coli and an important human pathogen, Mycobacterium tuberculosis have shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. My PhD thesis consists of four Chapters. Chapter 1 summarizes the relevant literature review on DNA damage and repair with an emphasis on uracil DNA glycosylase and its interacting protein, SSB. Chapters 2 and 3 describe my studies on the mechanism of uracil excision repair in E. coli. Chapter 4 describes my findings on the structure-function relationship of single stranded DNA binding proteins from E. coli and M. tuberculosis. Specific details of my research are summarized as follows: (1) Analysis of the impact of allelic exchange of ung with a mutant gene encoding Uracil DNA Glycosylase attenuated in AP-DNA binding in the maintenance of genomic integrity in Escherichia coli. There are five families of UDGs. Of these, Ung proteins (family 1 UDGs) represent highly efficient and evolutionary conserved enzymes. Structural and biochemical analysis of Ung proteins has identified two conserved motif, motif A (62GQDPY66) and motif B (187HPSPLS192) in E. coli that are important for the catalysis by Ung enzyme. Y66 of motif A is in van der Waals contact with the C5 position of the uracil and prevents entry of other bases. Earlier study from the laboratory showed that the Y66W and Y66H mutants of Ung were compromised by ~7 and ~170 fold, respectively in their uracil excision activities. However, unlike the wild-type and Y66H proteins, Y66W was not inhibited by its product (uracil or AP-DNA). In this study, by fluorescence anisotropy measurements I have shown that compared with the wild-type protein, the Y66W mutant is moderately compromised and attenuated in binding to AP-DNA. Allelic exchange of ung in E. coli with ung::kan, ungY66H:amp or ungY66W:amp alleles showed ~5, ~3.0 and ~2.0 fold, respectively increase in mutation frequencies. Analysis of mutations in the rifampicin resistance determining region (RRDR) of rpoB revealed that the Y66W allele resulted in an increase in A to G (or T to C) mutations. However, the increase in A to G mutations was mitigated upon expression of wild-type Ung from a plasmid borne gene. Biochemical and computational analyses showed that the Y66W mutant maintains strict specificity for uracil excision from DNA. Interestingly, a strain deficient in AP-endonucleases also showed an increase in A to G mutations. These findings have been discussed in the context of a proposal that the residency of DNA glycosylase(s) onto the AP-sites they generate shields them until recruitment of AP-endonucleases for further repair. It is proposed that an error prone replication against AP-sites (as a result of uracil excision activities on A:U pair) may result in A to G mutations. 2. Mechanism of appearance of A to G mutations in ungY66W:amp strain of Escherichia coli. In this part of my study, I have investigated the role of error prone DNA polymerases in the mutational specificity of ungY66W:amp strain. It was observed from various studies in E. coli that, DNA polymerase IV (Pol IV) and DNA polymerase V (Pol V) are involved in error-prone replication on damaged or AP-site containing DNA. E. coli strains containing deletion of either dinB (encoding DNA Pol IV) or umuDC (encoding DNA Pol V) were generated and used to study mutation frequency and mutation spectrum. Deletion of DNA Pol V resulted in a decrease in A to G mutations in ungY66W:amp E. coli strain, suggesting that increase in A to G mutations were a consequence of error prone incorporation by DNA Pol V. 3. Structure and Function studies on Single Stranded DNA Binding Proteins from Escherichia coli and Mycobacterium tuberculosis. SSB from M. tuberculosis (MtuSSB) has similar domain organization as the EcoSSB. Moreover, the biochemical properties such as oligomerization, DNA binding affinity and minimum binding site size requirements were shown to be similar to EcoSSB. However, structural studies suggested that quaternary structures of these two SSBs are variable. In this study I have used X-ray crystal structure information of these two SSBs to generate various chimeras after swapping at various regions of SSBs. Chimeras mβ1, mβ1’β2, mβ1-β5, mβ1-β6, and mβ4-β5 SSBs were generated by substituting β1 (residues 611), β1’β2 (residues 21-45), β1-β5 (residues 1 to 111), β1-β6 including a downstream sequence (residues 1 to 130), and β4-β5 (residues 74-111) regions of EcoSSB with the corresponding sequences of MtuSSB, respectively. Additionally, mβ1’β2ESWR SSB was generated by mutating the MtuSSB specific ‘PRIY’ sequence in the β2 strand of mβ1’β2 SSB to EcoSSB specific ‘ESWR’ sequence. Biochemical characterization revealed that except for mβ1 SSB, all chimeras and a control construct lacking the C-terminal domain (ΔC SSB) efficiently bound DNA in modes corresponding to limited and unlimited modes of binding. The mβ1 SSB was also hypersensitive to chymotrypsin treatment. The mβ1-β6, MtuSSB, mβ1’β2 and mβ1-β5 constructs complemented E. coli Δssb in a dose dependent manner. Complementation by the mβ1-β5 SSB was poor. In contrast, mβ1’β2ESWR SSB complemented E. coli as well as EcoSSB. Interestingly, the inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.

Les plantes doivent assurer la protection de trois génomes localisés dans le noyau, les chloroplastes et les mitochondries. Si les mécanismes assurant la réparation de l’ADN nucléaire sont relativement bien compris, il n’en va pas de même pour celui des chloroplastes et des mitochondries. Or il est important de bien comprendre ces mécanismes puisque des dommages à l’ADN non ou mal réparés peuvent entraîner des réarrangements dans les génomes. Chez les plantes, de tels réarrangements dans l’ADN mitochondrial ou dans l’ADN chloroplastique peuvent conduire à une perte de vigueur ou à un ralentissement de la croissance. Récemment, notre laboratoire a identifié une famille de protéines, les Whirly, dont les membres se localisent au niveau des mitochondries et des chloroplastes. Ces protéines forment des tétramères qui lient l’ADN monocaténaire et qui accomplissent de nombreuses fonctions associées au métabolisme de l’ADN. Chez Arabidopsis, deux de ces protéines ont été associées au maintien de la stabilité du génome du chloroplaste. On ignore cependant si ces protéines sont impliquées dans la réparation de l’ADN. Notre étude chez Arabidopsis démontre que des cassures bicaténaires de l’ADN sont prises en charge dans les mitochondries et les chloroplastes par une voie de réparation dépendant de très courtes séquences répétées (de cinq à cinquante paires de bases) d’ADN. Nous avons également montré que les protéines Whirly modulent cette voie de réparation. Plus précisément, leur rôle serait de promouvoir une réparation fidèle de l’ADN en empêchant la formation de réarrangements dans les génomes de ces organites. Pour comprendre comment les protéines Whirly sont impliquées dans ce processus, nous avons élucidé la structure cristalline d’un complexe Whirly-ADN. Nous avons ainsi pu montrer que les Whirly lient et protègent l’ADN monocaténaire sans spécificité de séquence. La liaison de l’ADN s’effectue entre les feuillets β de sous-unités contiguës du tétramère. Cette configuration maintient l’ADN sous une forme monocaténaire et empêche son appariement avec des acides nucléiques de séquence complémentaire. Ainsi, les protéines Whirly peuvent empêcher la formation de réarrangements et favoriser une réparation fidèle de l’ADN. Nous avons également montré que, lors de la liaison de très longues séquences d’ADN, les protéines Whirly peuvent s’agencer en superstructures d’hexamères de tétramères, formant ainsi des particules sphériques de douze nanomètres de diamètre. En particulier, nous avons pu démontrer l’importance d’un résidu lysine conservé chez les Whirly de plantes dans le maintien de la stabilité de ces superstructures, dans la liaison coopérative de l’ADN, ainsi que dans la réparation de l’ADN chez Arabidopsis. Globalement, notre étude amène de nouvelles connaissances quant aux mécanismes de réparation de l’ADN dans les organites de plantes ainsi que le rôle des protéines Whirly dans ce processus. / Plants must protect the integrity of three genomes located respectively in the nucleus, the chloroplasts and the mitochondria. Although DNA repair mechanisms in the nucleus are the subject of multiple studies, little attention has been paid to DNA repair mechanisms in chloroplasts and mitochondria. This is unfortunate since mutations in the chloroplast or the mitochondrial genome can lead to altered plant growth and development. Our laboratory has identified a new family of proteins, the Whirlies, whose members are located in plant mitochondria and chloroplasts. These proteins form tetramers that bind single-stranded DNA and play various roles associated with DNA metabolism. In Arabidopsis, two Whirly proteins maintain chloroplast genome stability. Whether or not these proteins are involved in DNA repair has so far not been investigated. Our studies in Arabidopsis demonstrate that DNA double-strand breaks are repaired in both mitochondria and chloroplasts through a microhomology-mediated repair pathway and indicate that Whirly proteins affect this pathway. In particular, the role of Whirly proteins would be to promote accurate repair of organelle DNA by preventing the repair of DNA double-strand breaks by the microhomology-dependant pathway. To understand how Whirly proteins mediate this function, we solved the crystal structure of Whirly-DNA complexes. These structures show that Whirly proteins bind single-stranded DNA with low sequence specificity. The DNA is maintained in an extended conformation between the β-sheets of adjacent protomers, thus preventing spurious annealing with a complementary strand. In turn, this prevents formation of DNA rearrangements and favors accurate DNA repair. We also show that upon binding long ssDNA sequences, Whirly proteins assemble into higher order structures, or hexamers of tetramers, thus forming spherical particles of twelve nanometers in diameter. We also demonstrate that a lysine residue conserved among plant Whirly proteins is important for the stability of these higher order structures as well as for cooperative binding to DNA and for DNA repair. Overall, our study elucidates some of the mechanisms of DNA repair in plant organelles as well as the roles of Whirly proteins in this process.

Les plantes doivent assurer la protection de trois génomes localisés dans le noyau, les chloroplastes et les mitochondries. Si les mécanismes assurant la réparation de l’ADN nucléaire sont relativement bien compris, il n’en va pas de même pour celui des chloroplastes et des mitochondries. Or il est important de bien comprendre ces mécanismes puisque des dommages à l’ADN non ou mal réparés peuvent entraîner des réarrangements dans les génomes. Chez les plantes, de tels réarrangements dans l’ADN mitochondrial ou dans l’ADN chloroplastique peuvent conduire à une perte de vigueur ou à un ralentissement de la croissance. Récemment, notre laboratoire a identifié une famille de protéines, les Whirly, dont les membres se localisent au niveau des mitochondries et des chloroplastes. Ces protéines forment des tétramères qui lient l’ADN monocaténaire et qui accomplissent de nombreuses fonctions associées au métabolisme de l’ADN. Chez Arabidopsis, deux de ces protéines ont été associées au maintien de la stabilité du génome du chloroplaste. On ignore cependant si ces protéines sont impliquées dans la réparation de l’ADN. Notre étude chez Arabidopsis démontre que des cassures bicaténaires de l’ADN sont prises en charge dans les mitochondries et les chloroplastes par une voie de réparation dépendant de très courtes séquences répétées (de cinq à cinquante paires de bases) d’ADN. Nous avons également montré que les protéines Whirly modulent cette voie de réparation. Plus précisément, leur rôle serait de promouvoir une réparation fidèle de l’ADN en empêchant la formation de réarrangements dans les génomes de ces organites. Pour comprendre comment les protéines Whirly sont impliquées dans ce processus, nous avons élucidé la structure cristalline d’un complexe Whirly-ADN. Nous avons ainsi pu montrer que les Whirly lient et protègent l’ADN monocaténaire sans spécificité de séquence. La liaison de l’ADN s’effectue entre les feuillets β de sous-unités contiguës du tétramère. Cette configuration maintient l’ADN sous une forme monocaténaire et empêche son appariement avec des acides nucléiques de séquence complémentaire. Ainsi, les protéines Whirly peuvent empêcher la formation de réarrangements et favoriser une réparation fidèle de l’ADN. Nous avons également montré que, lors de la liaison de très longues séquences d’ADN, les protéines Whirly peuvent s’agencer en superstructures d’hexamères de tétramères, formant ainsi des particules sphériques de douze nanomètres de diamètre. En particulier, nous avons pu démontrer l’importance d’un résidu lysine conservé chez les Whirly de plantes dans le maintien de la stabilité de ces superstructures, dans la liaison coopérative de l’ADN, ainsi que dans la réparation de l’ADN chez Arabidopsis. Globalement, notre étude amène de nouvelles connaissances quant aux mécanismes de réparation de l’ADN dans les organites de plantes ainsi que le rôle des protéines Whirly dans ce processus. / Plants must protect the integrity of three genomes located respectively in the nucleus, the chloroplasts and the mitochondria. Although DNA repair mechanisms in the nucleus are the subject of multiple studies, little attention has been paid to DNA repair mechanisms in chloroplasts and mitochondria. This is unfortunate since mutations in the chloroplast or the mitochondrial genome can lead to altered plant growth and development. Our laboratory has identified a new family of proteins, the Whirlies, whose members are located in plant mitochondria and chloroplasts. These proteins form tetramers that bind single-stranded DNA and play various roles associated with DNA metabolism. In Arabidopsis, two Whirly proteins maintain chloroplast genome stability. Whether or not these proteins are involved in DNA repair has so far not been investigated. Our studies in Arabidopsis demonstrate that DNA double-strand breaks are repaired in both mitochondria and chloroplasts through a microhomology-mediated repair pathway and indicate that Whirly proteins affect this pathway. In particular, the role of Whirly proteins would be to promote accurate repair of organelle DNA by preventing the repair of DNA double-strand breaks by the microhomology-dependant pathway. To understand how Whirly proteins mediate this function, we solved the crystal structure of Whirly-DNA complexes. These structures show that Whirly proteins bind single-stranded DNA with low sequence specificity. The DNA is maintained in an extended conformation between the β-sheets of adjacent protomers, thus preventing spurious annealing with a complementary strand. In turn, this prevents formation of DNA rearrangements and favors accurate DNA repair. We also show that upon binding long ssDNA sequences, Whirly proteins assemble into higher order structures, or hexamers of tetramers, thus forming spherical particles of twelve nanometers in diameter. We also demonstrate that a lysine residue conserved among plant Whirly proteins is important for the stability of these higher order structures as well as for cooperative binding to DNA and for DNA repair. Overall, our study elucidates some of the mechanisms of DNA repair in plant organelles as well as the roles of Whirly proteins in this process.