This is a pre-copyedited, author-produced PDF of an article accepted for publication in Mutagenesis following peer review. The definitive publisher-authenticated version is available online at:http://mutage.oxfordjournals.org/content/early/2015/08/03/mutage.gev055.full. / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19588号 / 医博第4095号 / 新制||医||1014(附属図書館) / 32624 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小泉 昭夫, 教授 渡邊 直樹, 教授 高田 穣 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Forensic scientists and ancient DNA researchers face similar challenges with respect to genetic information acquisition and analysis. However, these communities differ in one critical aspect: while forensic science is regulated by the strict guidelines of the judicial community, ancient DNA is a research-based academic field free to explore emerging technologies as they arise. This thesis investigates the application of two methodologies, developed in ancient DNA research, to challenging extracts, in hopes of modernizing forensic models while maintaining compatibility with current standards. The first chapter focuses on blunt-end sequencing library preparation protocols previously optimized for ancient DNA specimens. Forensically-relevant extracts were converted into libraries and typed by short tandem repeats (STR) amplification. When compared to STR profiles from pre-library extracts, a significant decrease in the quality was observed, in the form of allelic drop-out, heterozygous peak imbalance and increased stutter ratios. The second chapter discusses the efficacy of two enzymatic DNA repair methods, “PreCR® Repair” and “Nelson”, on typical ancient DNA specimens. Based on endogenous sample content, fragment length variation and base misincorporation rates, some DNA repair was reported when using PreCR®. However, the use of the Nelson protocol is not recommended for use in its current state. Both sequencing library preparation and enzymatic DNA repair show potential application to forensic evidential material, but require further analyses to confirm hypotheses and observations outlined in this thesis. / Thesis / Master of Science (MSc)

Fusarium oxysporum is a filamentous fungus that is known to invade over a hundred different hosts and poses a major threat to the economy and food supply world-wide. Poly (Adenosine diphosphate-Ribose) Polymerase (PARP) is a family of regulatory proteins that affect change in the cell through transfer of ADP-Ribose moieties onto target molecules. The most well-studied PARP protein is the human PARP1, a PARylating nuclear protein that serves as our model PARP protein. F. oxysporum was found to contain a large expansion of PARP catalytic-domain-containing proteins compared to other filamentous fungi. We utilized in silico multiple sequence alignments and domain predictions to identify a human PARP1 homolog termed foPARP1 that was conserved within the core chromosomes in all three strains within our comparative system. Our in silico predictions also stated that only one strain, an Arabidopsis pathogen, Fo5176, contained several other predicted catalytically active PARP homologs within the accessory chromosome. To test the effect that foPARP1 knockout would have on DNA damage tolerance, we created a foParp1 knockout and found that only strains Fol4287 and Fo5176 had a significant reduction in tolerance upon being plated with methyl methanesulfonate (MMS), a DNA alkylating agent. To test how global PARylation trends would be affected by foParp1 knockout, we utilized immunodot-blotting with PAR antibodies to assess PARylation in total protein extracts. We found that all strains of the comparative system had the capacity to catalyze the synthesis of long PAR chains, while only Fo47 and Fo5176 had a significant PARylation increase when exposed to MMS, and no samples had a significant increase in PARylation within the foParp1 knockouts. Finally, we utilized RNA-Sequencing to determine the transcriptional impacts that foParp1 knockout would have and found aberrant DNA repair pathways and disruptions in stress responses. Taken together, we conclude that foPARP1 is in fact a functional PARP1 homolog and exhibits similar post-transcriptional modification and transcriptional impacts as its human counterpart. However, we were not able to correlate PARP copy number with DNA stress tolerance, and further research would be needed to assess the full function of the PARP expansion.

TRF1, telomeric-repeat binding factor 1, is a component of the six-subunit protein complex, referred to as shelterin, which is essential for not only regulating telomere length maintenance but also protecting mammalian telomeres from being recognized as damaged DNA. TRF1 acts as a negative mediator of telomerase-dependent telomere elongation in telomerase-expressing cells, whereas it promotes alternative lengthening of telomeres (ALT) activity by regulating ALT features including the production of extrachromosomal telomere-repeat (ECTR) DNA such as C-circles, and ALT-associated promyelocytic leukemia bodies, or APBs. The activity of TRF1 is tightly regulated by post-translational modification such as phosphorylation. This thesis sets out to investigate the function of TRF1 phosphorylation on threonine-271 (T271) and threonine-371 in telomere maintenance. The results presented in this thesis demonstrate that TRF1 phosphorylation on T271 positively regulates the association of TRF1 to telomeric DNA in telomerase expressing cells. In ALT cells, TRF1 phosphorylation on both T271 and T371 is shown to be important for the formation of APBs. Furthermore, the work presented here suggests that transcription-associated DNA damage mediates the association of phosphorylated (pT371)TRF1 with APBs. / Thesis / Master of Science (MSc) / TRF1, telomeric-repeat binding factor 1, is a component of the shelterin complex, which is essential for regulating telomere length maintenance and protecting mammalian telomeres from being recognized as damaged DNA. TRF1 acts as a negative mediator of telomerase-dependent telomere elongation in telomerase-expressing cells, whereas it promotes alternative lengthening of telomeres. The activity of TRF1 is tightly regulated by phosphorylation. This thesis sets out to investigate the function of TRF1 phosphorylation on threonine-271 and threonine-371 in telomere maintenance. Understanding how post-translational modifications on TRF1 may be linked to telomere homeostasis will be crucial for our understanding in cancer cell biology.

Translesion DNA Synthesis (TLS) is a mechanism that promotes DNA damage tolerance during DNA replication using an error-prone DNA polymerase complex. The complex is comprised of the ImuA, ImuB, and ImuC proteins that are found in approximately one-third of bacteria, including high priority antimicrobial resistant pathogens, such as Pseudomonas aeruginosa. Previous in vivo studies have shown that TLS increases beneficial bacterial mutations as the error-prone DNA polymerase, ImuC, lacks proof-reading activity. However, how ImuA and ImuB proteins contribute to the polymerase mechanism is unknown. Thus, the goal of this study is to characterize the TLS proteins in vitro to determine how ImuA and ImuB associate with ImuC to promote error- prone replication. ImuA and ImuBNΔ34 were successfully purified for biochemical characterization from the homolog Myxococcus xanthus. Using size-exclusion chromatography coupled to multi-angle light scattering, both ImuA and ImuBNΔ34 are trimers in solution. Each protein also binds DNA independently as assessed by fluorescence polarization. Interestingly, both proteins bind ssDNA and a 3’ overhang substrate mimicking the DNA replication intermediate with the highest affinity. DNA binding assays further confirm these proteins can form a DNA-ImuA-ImuBNΔ34 complex. Using bacterial two-hybrid assays, the ImuA- ImuB interaction occurs in the C-terminal region of both proteins. Overall, these results suggest that ImuA and ImuB may recruit and stabilize ImuC on DNA for replication past damaged DNA, providing the first insights into the ImuA and ImuB molecular mechanism. / Thesis / Master of Science (MSc)

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