The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing. Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension. The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results. Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses. Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results. <b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis

<p>The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing.</p><p>Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension.</p><p>The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results.</p><p>Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses.</p><p>Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results.</p><p><b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis</p>

For survival and successful propagation, every organism has to maintain the genomic integrity of the cell. The information content, in the form of nucleotide bases, is constantly threatened by endogenous agents and environmental pollutants. In particular, pathogenic mycobacteria are constantly exposed to DNA-damaging assaults such as reactive oxygen species (ROS) and reactive nitrogen intermediate (RNI), in their habitat which is inside host macrophage. In addition, the genome of Mycobacterium tuberculosis makes it more susceptible for guanine oxidation and cytosine deamination as it is G-C rich. Therefore DNA repair mechanisms are extremely important for the mycobacterium. An important enzyme involved in DNA repair is uracil-DNA glycosylase (Ung). To access the genomic information, during repair as well as DNA replication and recombination, dsDNA must unwind to form single stranded (ss) intermediates. ssDNA is more prone to chemical and nuclease attacks that can produce breaks or lesions and can also inappropriately self associate. In order to preserve ssDNA intermediates, cells have evolved a specialized class of ssDNA-binding proteins (SSB) that associate with ssDNA with high affinity. As part of a major programme on mycobacterial proteins in this laboratory, structural studies on mycobacterial uracil-DNA glycosylase (Ung) and single-stranded DNA binding protein (SSB) have been carried out. The structures were solved using the well-established techniques of protein X-ray crystallography. The hanging drop vapour diffusion and microbatch methods were 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. The data were processed using the HKL program suite. The structures were solved by the molecular replacement method using the program PHASER and AMoRe. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using COOT. PROCHECK, ALIGN, INSIGHT and NACCESS were used for structure validation and analysis of the refined structures. MD simulations were performed using the software package GROMACS v 3.3.1. Uracil-DNA glycosylase (UNG), a repair enzyme involved in the excision of uracil from DNA, from mycobacteria differs from UNGs from other sources, particularly in the sequence in the catalytically important loops. The structure of the enzyme from Mycobacterium tuberculosis (MtUng) in complex with a proteinaceous inhibitor (Ugi) has been determined by X-ray analysis of a crystal containing seven crystallographically independent copies of the complex. This structure provides the first geometric characterization of a mycobacterial UNG. A comparison of the structure with those of other UNG proteins of known structure shows that a central core region of the molecule is relatively invariant in structure and sequence, while the N- and C-terminal tails exhibit high variability. The tails are probably important in folding and stability. The mycobacterial enzyme exhibits differences in UNG-Ugi interactions compared with those involving UNG from other sources. The MtUng-DNA complex modelled on the basis of the known structure of the complex involving the human enzyme indicates a domain closure in the enzyme when binding to DNA. The binding involves a larger burial of surface area than is observed in binding by human UNG. The DNA-binding site of MtUng is characterized by the presence of a higher proportion of arginyl residues than is found in the binding site of any other UNG of known structure. In addition to the electrostatic effects produced by the arginyl residues, the hydrogen bonds in which they are involved compensate for the loss of some interactions arising from changes in amino-acid residues, particularly in the catalytic loops. The results arising from the present investigation represent unique features of the structure and interaction of mycobacterial Ungs. To gain further insights, the structure of Mycobacterium tuberculosis Ung (MtUng) in its free form was also determined. Comparison with appropriate structures indicate that the two domain enzyme slightly closes up when binding to DNA while it slightly opens up when binding to its proteinaceous inhibitor Ugi. The structural changes on complexation in the catalytic loops reflect the special features of their structure in the mycobacterial protein. A comparative analysis of available sequences of the enzyme from different sources indicates high conservation of amino acid residues in the catalytic loops. The uracil binding pocket in the structure is occupied by a citrate ion. The interactions of the citrate ion with the protein mimic those of uracil in addition to providing insights into other possible interactions that inhibitors could be involved in. SSB is an essential accessory protein required during DNA replication, repair and recombination, and various other DNA transactions. Eubacteral single stranded DNA binding (SSB) proteins constitute an extensively studied family of proteins. The variability in the quaternary association in these tetrameric proteins was first demonstrated through the X-ray analysis of the crystal structure of Mycobacterium tuberculosis SSB (MtSSB) and Mycobacterium smegmatis (MsSSB) in this laboratory. Subsequent studies on these proteins elsewhere have further explored this variability, but attention was solely concentrated on the variability in the relative orientation of the two dimers that constitute the tetramer. Furthermore, the effect of this variability on the properties of the tetrameric molecule was not adequately addressed. In order to further explore this variability and strengthen structural information on mycobacterial SSBs in particular, and on SSB proteins in general, the crystal structures of two forms of Mycobacterium leprae single stranded DNA-binding protein (MlSSB) has been determined. Comparison of the structures with other eubacterial SSB structures indicates considerable variation in their quaternary association although the DNA binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but it appears to correlate well with variation in protein stability. Molecular dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype E. coli SSB and the individual subunits of both the proteins. The X-ray studies and molecular dynamics simulations together yield information on the relatively rigid and flexible regions of the molecule and the effect of oligomerization on flexibility. The simulations provide insights into the changes in the subunit structure on oligomerization. They also provide insights into the stability and time evolution of the hydrogen bonds/water-bridges that connect two pairs of monomers in the tetramer. In continuation of our effort to understand structure-function relationships of mycobacterial SSBs, the structure of MsSSB complexed with a 31-mer polydeoxy-cytidine single stranded DNA (ssDNA) was determined. The mode of ssDNA binding in the MsSSB is different from the modes in the known structures of similar complexes of the proteins from E. coli (EcSSB) and Helicobacter pylori (HpSSB). The modes in the EcSSB and HpSSB also exhibit considerable differences between them. A comparison of the three structures reveals the promiscuity of DNA-binding to SSBs from different species in terms of symmetry and the path followed by the bound DNA chain. It also reveals commonalities within the diversity. The regions of the protein molecule involved in DNA-binding and the nature of the residues which interact with the DNA, exhibit substantial similarities. The regions which exhibit similarities are on the central core of the subunit which is unaffected by tetramerisation. The variable features of DNA binding are associated with the periphery of the subunit, which is involved in oligomerization. Thus, there is some correlation between variability in DNA-binding and the known variability in tetrameric association in SSBs. In addition to the work on Ung and SSB, the author was involved in X-ray studies on crystals of horse methemoglobin at different levels of hydration, which is described in the Appendix of the thesis. The crystal structure of high-salt horse methaemoglobin has been determined at environmental relative humidities (r.h.) of 88, 79, 75 and 66%. The molecule is in the R state in the native and the r.h. 88% crystals. At r.h.79% the molecule appears to move towards the R2 state. The crystal structure at r.h.66% is similar, but not identical, to that at r.h.75%. Thus variation in hydration leads to variation in the quaternary structure. Furthermore, partial dehydration appears to shift the structure from the R state to the R2 state. This observation is in agreement with the earlier conclusion that the changes in protein structure that accompany partial dehydration are similar to those that occur during protein action. A part of the work presented in the thesis has been reported in the following publications. 1. Singh, P., Talawar, R.K., Krishna, P.D., Varshney, U. & Vijayan, M. (2006). Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor. Acta Crystallogr. F62, 1231-1234. 2. Kaushal, P.S., Talawar, R.K., Krishna, P.D., Varshney, U. & Vijayan, M. (2008). Unique features of the structure and interactions of mycobacterial uracil-DNA glycosylase: structure of a complex of the Mycobacterium tuberculosis enzyme in comparison with those from other sources. Acta Crystallogr. D64, 551-560. 3. Kaushal, P.S., Sankaranarayanan, R. & Vijayan, M. (2008). Water-mediated variability in the structure of relaxed-state haemoglobin. Acta Crystallogr. F64, 463-469.

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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.

Mutations in the hereditary breast/ovarian cancer genes BRCA1/2 were shown to be synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). This toxicity is assumed to derive from PARPi-induced DNA double strand breaks (DSBs) that necessitate BRCA function in homologous recombination (HR) and/or fork protection (FP). However, PARPi accelerates replication forks. While high-speed replication could cause DSBs, the finding that PARPi leads to single-stranded DNA (ssDNA) gaps/nicks suggests replication gaps could also or alone be the cause of synthetic lethality. Here, we demonstrate that PARPi toxicity derives from replication gaps. Isogenic cells deficient in BRCA1 or the BRCA1-associated FANCJ, with common DNA repair defects in HR and FP, exhibit opposite responses to PARPi. Deficiency in FANCJ, a helicase also mutated in hereditary breast/ovarian cancer and Fanconi anemia, causes aberrant accumulation of fork remodeling factor HLTF and limits unrestrained DNA synthesis with ssDNA gaps. Thus, we predict replication gaps as a distinguishing factor and further uncouple HR, FP and fork speed from PARPi response. BRCA-deficient cells display excessive gaps that are diminished upon resistance, restored upon re-sensitization and when targeted augment synthetic lethality with PARPi. Furthermore, we define the source of gaps to defects in Okazaki fragment processing (OFP). Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1 but aberrantly low XRCC1 indicating a defective backup OFP pathway. Remarkably, 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. Collectively, our study highlights unprotected lagging strand gaps as a determinant of synthetic lethality, providing a new paradigm and biomarker for PARPi toxicity.

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.