Deficiency in Msh2, a component of the mismatch repair (MMR) system, leads to a ~10-fold increase in the mutation frequency in most tissues. By contrast, Msh2-deficiency in germinal center (GC) B cells decreases the mutation frequency at the IgH V-region, as a dU:dG mismatch produced by AID initiates modifications by MMR resulting in mutations at nearby A:T basepairs. This raises the possibility that GC B cells express a factor that converts MMR into a globally mutagenic pathway. To test this notion, we investigated whether MMR corrects mutations in GC B cells at a gene not mutated by AID. We found that GC B cells accumulate 5-times more mutations than follicular B cells. Notably, the mutation frequency was ~10 times higher in Msh2-/- compared to wildtype GC B cells. These results show that in GC B cells MMR functions normally at an AID-insensitive gene.

The processes of secondary antibody diversification are initiated by the mutagenic, B cell specific enzyme, Activation-Induced Deaminase (AID). AID deaminates deoxycytosine (dC) that is located in single-stranded DNA (ssDNA) in actively transcribed DNA to initiate the processes of somatic hypermutation (SHM), gene conversion (GCV) and class switch recombination (CSR) at the antibody gene loci. These processes lead to high affinity antibodies and antibodies of various effector functions that are required to efficiently neutralize invading pathogens. It is currently unclear how the antibody genes are specifically targeted by AID over other genes. I found that AID is able to mutate a non-immunoglobulin (Ig) transgene independent of its chromosomal integration site at rates that were above background mutation rates, but were ~10-fold lower than at the antibody variable (V) region. This result suggests that AID can mutate non-Ig genes at low rates, which may explain AID’s role in oncogenesis, but nevertheless shows that AID preferentially mutates the Ig locus over other loci. While it is understood that AID specifically deaminates dC bases in ssDNA, the size, distribution and origin of these ssDNA substrates is unknown. By utilizing a unique in situ sodium bisulfite assay to detect regions of ssDNA in intact nuclei, I characterized ssDNA regions and found that they are accurate predictors of AID activity during the processes of SHM and CSR in mammalian B cells and E.coli. Importantly, with the use of E.coli models, I show that these ssDNA substrates are the product of transcription-induced negative-supercoiled DNA that correlates strongly with the mutagenic activity of AID. While several underlying mechanisms exist to prevent the mistargeting of AID, my findings suggest that by simply gaining access to ssDNA that is produced by transcription-induced negative supercoiling, AID has the potential to mutate non-Ig genes, albeit at lower rates than the antibody V-region.

The processes of secondary antibody diversification are initiated by the mutagenic, B cell specific enzyme, Activation-Induced Deaminase (AID). AID deaminates deoxycytosine (dC) that is located in single-stranded DNA (ssDNA) in actively transcribed DNA to initiate the processes of somatic hypermutation (SHM), gene conversion (GCV) and class switch recombination (CSR) at the antibody gene loci. These processes lead to high affinity antibodies and antibodies of various effector functions that are required to efficiently neutralize invading pathogens. It is currently unclear how the antibody genes are specifically targeted by AID over other genes. I found that AID is able to mutate a non-immunoglobulin (Ig) transgene independent of its chromosomal integration site at rates that were above background mutation rates, but were ~10-fold lower than at the antibody variable (V) region. This result suggests that AID can mutate non-Ig genes at low rates, which may explain AID’s role in oncogenesis, but nevertheless shows that AID preferentially mutates the Ig locus over other loci. While it is understood that AID specifically deaminates dC bases in ssDNA, the size, distribution and origin of these ssDNA substrates is unknown. By utilizing a unique in situ sodium bisulfite assay to detect regions of ssDNA in intact nuclei, I characterized ssDNA regions and found that they are accurate predictors of AID activity during the processes of SHM and CSR in mammalian B cells and E.coli. Importantly, with the use of E.coli models, I show that these ssDNA substrates are the product of transcription-induced negative-supercoiled DNA that correlates strongly with the mutagenic activity of AID. While several underlying mechanisms exist to prevent the mistargeting of AID, my findings suggest that by simply gaining access to ssDNA that is produced by transcription-induced negative supercoiling, AID has the potential to mutate non-Ig genes, albeit at lower rates than the antibody V-region.

abstract: The communication of genetic material with biomolecules has been a major interest in cancer biology research for decades. Among its different levels of involvement, DNA is known to be a target of several antitumor agents. Additionally, tissue specific interaction between macromolecules such as proteins and structurally important regions of DNA has been reported to define the onset of certain types of cancers. Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are discussed. Chapter 2 describes a new approach to study the double-strand DNA lesion caused by antitumor drug bleomycin. The hairpin DNA library used in this study displays numerous cleavage sites demonstrating the versatility of bleomycin interaction with DNA. Interestingly, some of those cleavage sites suggest a novel mechanism of bleomycin interaction, which has not been reported before. Cytidine methylation has generally been found to decrease site-specific cleavage of DNA by BLM, possibly due to structural change and subsequent reduced bleomycin-mediated recognition of DNA. As illustrated in Chapter 3, three hairpin DNAs known to be strongly bound by bleomycin, and their methylated counterparts, were used to study the dynamics of bleomycin-induced degradation of DNAs in cancer cells. Interestingly, cytidine methylation on one of the DNAs has also shown a major shift in the intensity of bleomycin induced double-strand DNA cleavage pattern, which is known to be a more potent form of bleomycin induced cleavages. DNA secondary structures are known to play important roles in gene regulation. Chapter 4 demonstrates a structural change of the BCL2 promoter element as a result of its dynamic interaction with the individual domains of hnRNP LL, which is essential to facilitate the transcription of BCL2. Furthermore, an in vitro protein synthesis technique has been employed to study the dynamic interaction between protein domains and the i-motif DNA within the promoter element. Several constructs were made involving replacement of a single amino acid with a fluorescent analogue, and these were used to study FRET between domain 1 and the i-motif, the later of which harbored a fluorescent acceptor nucleotide analogue. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2014

Les protéines APOBEC3 (A3A-A3H) catalysent la désamination des cytidines (C) présentes sur l'ADN simple brin en thymidine (T). Cette activité cytidines désaminase a initialement été décrite comme impliquée dans la restriction des rétrovirus et de certains virus à ADN par leur capacité à induire de nombreuses mutations C->T, ou hypermutations, sur les génomes viraux. Il apparait néanmoins que leur activité n'est pas restreinte aux génomes viraux et que certaines A3 peuvent induire des mutations sur l'ADN mitochondrial (A3A, C, F, G et H) et nucléaire (A3A et A3B). Ainsi, l'impact somatique des A3 est désormais établi dans la formation de certains cancers, dont la majorité des mutations, portent signatures des APOBEC3. Aux vues de ces observations, nous nous sommes intéressés à la façon dont sont régulées ces enzymes dans le contexte du stress cellulaire viro-induit ou endogène. La première partie de nos travaux a porté sur la protéine A3DE, seul membre de la famille APOBEC3 ne possédant pas d'activité cytidine désaminase. De façon intéressante, il apparait qu'A3DE est surexprimée dans les cirrhoses infectées par le VHB, VHC ou co-infectées par le VHC et le VHB. Nous avons pu mettre en évidence qu'A3DE interagit et module l'activité d'A3F et d'A3G, deux cytidines désaminases exprimées dans le foie et impliquées dans la restriction du VHB. Dans un second temps, nous nous sommes intéressés à la caractérisation du potentiel génotoxique de la protéine A3B. Cette protéine, de par sa localisation strictement nucléaire, constitue la seule A3 à double domaine n'interagissant pas avec A3DE. Contrairement à A3A, A3B est faiblement active sur l’ADN nucléaire et n’induit pas de cassures de l’ADN double brin. Nous avons pu mettre en évidence par mutagénèse les régions de la protéine impliquées dans l’atténuation de la génotoxicité d’A3B par rapport à A3A et que cette atténuation est conservée chez les primates. Enfin, nous avons étudié le rôle et la régulation d’A3A dans le catabolisme. Nous avons mis en évidence que l’ADN mitochondrial cytoplasmique (ADNcymt) active la voie RIG-I/ARN polymérase III ce qui a pour effet d’induire la production d’IFN qui va activer l’expression d’A3A. A3A va ainsi jouer un rôle dans le catabolisme de l’ADNcymt et contribue à l'élimination de cette source de stress cellulaire, mais occasionnant par la même des dommages sur l’ADN nucléaire. Les A3 sont des enzymes fondamentales de la défense immunitaire innée et du catabolisme de l’ADN. Nous montrons qu’A3DE a pour fonction de moduler l’activité d’A3F et d’A3G tandis qu’A3B, possède un phénotype atténué chez tous les primates et s’avère moins génotoxique que’A3A. Cette dernière participe à la dégradation de l’ADN cytoplasmique, limitant ainsi l’inflammation. Néanmoins, A3A peut s’avérer dangereuse pour l’intégrité génomique et contribuer à l’émergence de cancers, notamment en cas d’inflammation chronique. / APOBEC3 proteins (A3A-A3H) catalyse the deamination of cytosine (C) to thymidine (T) on single stranded DNA. This activity, called cytidine deaminase, has initially been described as a mechanism involved in restriction against retroviruses and DNA viruses by massively inducing C->T mutations on viral genome : this phenomenon is called "hypermutations". Nevertheless, this activity is not virus-specific and some A3 can induce mutations on mitochondrial DNA (A3A, C, F, G, H) and nuclear DNA (A3A and A3B). Thus, the impact of those proteins on cancer formation is now established in cancers where mutations mostly show an APOBEC3 signature. In view of those considerations, we decided to study how those enzymes are regulated in the context of a viral cellular stress or an endogenous cellular stress. The first part of our work is focused on A3DE, the only APOBEC3 lacking a cytidine deaminase activity. Interestingly, A3DE is upregulated in cirrhotic livers infected by HBV, HCV or coinfected with HBV & HCV. We show that A3DE inhibits A3F & A3G activity by interacting with those HBV restriction involved A3. Then, we studied the attributes of the genotoxicity potential of A3B. This protein, by his strictly nuclear localization, constitutes the only double domain A3 which is not regulated by A3DE. Unlike A3A, A3B is weakly active on nuclear DNA and does not induce double strand breaks. We determine by directed mutagenesis the clusters of A3B involved in genotoxicity attenuation compared with A3A. We also show that this attenuation is conserved among primates. Finally, we investigated the role and regulation of A3A in the context of DNA catabolism. We proved that mitochondrial cytoplasmic DNA (mtcyDNA) triggers the RIG-I/DNA polymerase III pathway, which induces IFN production leading to A3A expression. So A3A will be involved in mtcyDNA catabolism and contribute to the clearance of this stress signal, but will also induce double strand breaks on nuclear DNA. A3 are major enzymes of the innate immune response and DNA catabolism. We show that A3DE modulates A3F and A3G activity while A3B is attenuated among primates and is less genotoxic than A3A. A3A participates to cytoplasmic DNA catabolism and limits inflammation. Nevertheless, A3A could be dangerous for the genomic integrity and contributes to cancer, especially in cases of chronic inflammation.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22698号 / 医科博第113号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 竹内 理, 教授 椛島 健治, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

This dissertation is author version of following the journal article. Takahiro Shimizu, Hiroyuki Marusawa, Yuko Matsumoto, Tadashi Inuzuka, Atsuyuki Ikeda, Yosuke Fujii, Sachiko Minamiguchi, Shin’ichi Miyamoto, Tadayuki Kou, Yoshiharu Sakai, Jean E. Crabtree, Tsutomu Chiba, Accumulation of Somatic Mutations in TP53 in Gastric Epithelium With Helicobacter pylori Infection, Gastroenterology, Volume 147, Issue 2, August 2014, Pages 407-417.e3, ISSN 0016-5085, http://dx.doi.org/10.1053/j.gastro.2014.04.036. / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18543号 / 医博第3936号 / 新制||医||1006(附属図書館) / 31443 / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 小川 誠司, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19228号 / 医博第4027号 / 新制||医||1011(附属図書館) / 32227 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武田 俊一, 教授 竹内 理, 教授 髙田 穣 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19567号 / 医博第4074号 / 新制||医||1013(附属図書館) / 32603 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武田 俊一, 教授 小川 誠司, 教授 野田 亮 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24833号 / 医博第5001号 / 新制||医||1067(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田, 宏一, 教授 上野, 英樹, 教授 濵﨑, 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM