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The role of DNA polymerase β, one of main factors in base excision repair, in the nervous system
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 이영수 | - |
| dc.contributor.author | 김주식 | - |
| dc.date.accessioned | 2022-11-29T02:32:14Z | - |
| dc.date.available | 2022-11-29T02:32:14Z | - |
| dc.date.issued | 2020-08 | - |
| dc.identifier.other | 30312 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/19742 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2020. 8 | - |
| dc.description.tableofcontents | I. INTRODUCTUON 1 A. Base excision repair and DNA single strand break repair 1 B. X–ray repair cross complementing 1 2 C. DNA polymerase β 4 D. Hereditary neurodegenerative disease related single strand break in nervous system 4 E. The mouse models of XRCC1–binding proteins 5 F. DNA damage response 6 G. The structure and function of ATM and A–T 7 H. The problem of ATM animal models 7 I. Genomic instability and cancer 8 J. Medulloblastoma 9 K. Hypothesis and specific aims 10 II.MATERIALS AND METHODS 21 A. Animals 21 B. Quantitative realtime PCR 22 C. Western blot analysis 25 D. Histopathological analysis 26 E. Genomics analysis 27 F. Enzyme–linked immunosorbent assay (ELISA) and chemiluminescent assay 30 G. Image and statistical analyses 30 III. RESULTS 32 A. Polb inactivation during neurogenesis results in the loss of cerebral cortical interneuron without noticeable neurodevelopmental defects 32 B. Polb inactivation leads to DNA damage and ATM dependent apoptosis in the ganglionic eminence during neurogenesis 37 C. Double inactivation of Polb and Atm in the nervous system results in severe ataxia 42 D. Inactivation of Xrcc1 and Atm during neurogenesis results in cerebellar neurodevelopmental defect, yet Polb and Atm double inactivation does not 47 E. Itpr1 is the most differentially expressed gene in PolbNes–CreAtm–/– cerebella 52 F. Itpr1 expression is reduced progressively toward cerebellar ataxia 57 G. The total level of cytosine methylation is not different in the Polb/Atm and Xrcc1/Atm double knockout cerebella 61 H. Altered cytosine demethylation could result in Itpr1 reduction in the Polb/Atm and Xrcc1/Atm double knockout cerebella 66 I. TET1 activity is reduced in the Polb/Atm and Xrcc1/Atm double knockout cerebella 71 J. Polb/p53 double null animals develop medulloblastoma 76 K. DNA damage is accumulated in Polb/p53 and Xrcc1/p53 double null medulloblastomas 79 L. Polb/p53 and Xrcc1/p53 double null medulloblastomas show SHHα subgroup gene expression 82 IV. DISCUSSION 85 REFERENCE 94 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | The role of DNA polymerase β, one of main factors in base excision repair, in the nervous system | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.department | 일반대학원 의생명과학과 | - |
| dc.date.awarded | 2020. 8 | - |
| dc.description.degree | Doctoral | - |
| dc.identifier.localId | 1151653 | - |
| dc.identifier.uci | I804:41038-000000030312 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/common/orgView/000000030312 | - |
| dc.subject.keyword | ATM | - |
| dc.subject.keyword | Cerebellar ataxia | - |
| dc.subject.keyword | Medulloblastoma | - |
| dc.subject.keyword | POLB | - |
| dc.subject.keyword | p53 | - |
| dc.description.alternativeAbstract | Base excision repair pathway plays important roles in repairing damaged DNA bases or removing chemically modified DNA bases during DNA demethylation process for gene expression. POLB is one of key enzymes for this DNA repair pathway, which acts as a DNA lyase and DNA polymerase at DNA damage sites through binding to XRCC1. XRCC1 is a scaffolding protein and has a crucial role for recruiting several DNA repair proteins to the sites of damaged bases or strand breaks. Human XRCC1 mutations are responsible for the spinocerebellar ataxia 26 and the deletion of the Xrcc1 gene in the murine nervous system led to several neurological phenotypes including the loss of cerebellar interneurons. Interestingly, the severe cerebellar ataxia due to abnormal cerebellar development was observed in the Xrcc1/Atm double null animal model. A protein kinase ATM responds to DNA strand breaks, and mutations in ATM are responsible for Ataxia Telangiectasia (A–T), which is characterized by progressive cerebellar ataxia. It is not fully understood yet the molecular mechanisms of the DNA damage repair defects and cerebellar ataxia, although several human genetic diseases as exemplified above indicate the strong connection between those two. So the working hypothesis was POLB is the responsible protein for the XRCC1 related neurologic phenotypes. To study the connection between POLB and XRCC1 in the nervous system, an animal model of selective Polb inactivation during neurogenesis was generated and examined. In contrast to Xrcc1 deficiency in the murine nervous system, inactivation of Polb affected only a subpopulation of cortical intereurons despite the accumulation of DNA damage throughout the brain, and there was no sign of cerebellar interneuron loss. The dual inactivation of Polb and Atm resulted in cerebellar ataxia without significant neuropathological defects, which was different from the Xrcc1 and Atm inactivated animal model. In the cerebella of mice deficient for both Polb and Atm, the most downregulated gene was Itpr1, likely because of misregulated DNA methylation cycle, in which POLB is involved, and reduced enzyme activity of methylcytosine hydroxylase TET1 that could be activated by ATM upon DNA damage. ITPR1 is known to mediate calcium homeostasis, and ITPR1 mutations result in genetic diseases with cerebellar ataxia. These finding suggest defective calcium homeostasis due to dysregulation of ITPR1 in the cerebellum could be one of contributing factors to progressive ataxia observed in human genomic instability syndromes. Furthermore, similarly to the Xrcc1 and p53 double null animal model, which develops medulloblastoma, the Polb/p53 animal model also develop the brain tumor. Medulloblastoma, which occurs in the cerebellum with a high incidence in children, is divided into four subgroups dependent on the molecular expression profiles and its origin (WNT, SHH, Group 3, and Group 4). The medulloblastoma due to inactivation of either Polb or Xrcc1 in deletion of p53 background belonged to the SHH type, particularly SHHα subtype verified by the representative gene expressions for that subtype, such as Atoh1, Gli2, Ptch2, and Sfrp1, suggesting that granule cell origin of the medulloblastoma, also underscores the important role of genomic stability in preventing this devastating pediatric cerebellar tumor. My data suggest that POLB is important for cerebral interneuron genesis and cerebellar function by preventing accumulation of endogenous DNA damage and proper DNA methylation cycle. | - |
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