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Characterization of Mec1p/Tel1p-dependent Cdc13p phosphorylation and non-homologous end-joining mechanism in Saccharomyces cerevisiae.
No full text藉由Mec1p與Tel1p磷酸化調控端粒附著蛋白Cdc13的端粒酶吸引區域
DNA損傷反應蛋白激酶ATM與ATR在生體內(in vivo)大部份它們所磷酸化的對象都是SQ/TQ聚集在一起的序列。在出芽酵母菌中的蛋白Cdc13p具有兩個SQ/TQ聚集在一起的序列暗示著它可能可以被Mec1p與Tel1p(酵母菌中的ATM與ATR)磷酸化。在這篇研究中我們證明了Cdc13p中吸引端粒酶區域可以被Mec1p與Tel1p磷酸化。利用膠條分析方式證明Cdc13p包含了依賴Mec1p與Tel1p的轉譯後修飾。使用免疫沉澱-激酶分析我們證明了在生體外(in vitro)Mec1p可以磷酸化Cdc13p中絲胺酸225、249、255與306的位置而Tel1p則可以磷酸化絲胺酸225、249和255的位置。在生體內的表現型分析顯示出將Cdc13p中可以被Mec1p與Tel1p磷酸化的SQ序列突變會造成很多端粒及生長分面的缺陷。另外，表現Cdc13-Est1p融合蛋白在那些突變株中可以恢復正常的端粒長度及生長速度。這些結果證明Cdc13p中的端粒酶吸引區域是Mec1p及Tel1p的一個重要且新的端粒專一性的對像。
酵母菌中DNA聚合酶α、β、δ、ε和γ均參與在非同源性末端連結機制
大部分的DNA修補機制中都需要DNA聚合酶來執行核酸的修補延長。非同源性末端連結(NHEJ)參與在染色體雙股DNA斷裂修補機制中及B淋巴細胞與T淋巴細胞發育時抗原結合基多樣區域的連結。然而，在生體內(in vivo)有哪些DNA聚合酶透過NHEJ的方式來修補DNA雙股斷裂目前仍然不清楚。NHEJ修補DNA的方式包含了簡單末端連結(simple end-joining)及染色體重組(chromosomal rearrangement)。在這篇研究我們在酵母菌系統中使用生體內分析方式來探討不同的DNA聚合酶在簡單末端連結與染色體重組中所扮演的角色。本篇研究包含了酵母菌中六個主要的DNA聚合酶 (α、β、γ、δ、ε和ζ)。DNA聚合酶β被發現參與在簡單末端連結中加鹼基的步驟並且被證明它會被吸引到DNA雙股斷裂的位置。DNA聚合酶ε則被發現透過它的3’到5’外切核酸酶的活性參與在NHEJ中簡單末端連結中減鹼基的步驟。並且發現DNA聚合酶δ與γ都參與在染色體重組但不參與在簡單末端連結的機制中。總而言之，我們的研究證明了大部分的DNA聚合酶都參與在NHEJ的機制中，並且個別在細步的NHEJ機制中扮演不同的角色。The telomerase-recruitment domain of the telomere binding protein Cdc13 is regulated by Mec1p/Tel1p-dependent phosphorylation
The DNA damage-responsive protein kinases ATM and ATR phosphorylate SQ/TQ motifs that lie in clusters in most of their in vivo targets. Budding yeast Cdc13p contains two clusters of SQ/TQ motifs, suggesting that it might be a target of Mec1p/Tel1p (yeast ATR/ATM). Here we demonstrated that the telomerase-recruitment domain of Cdc13p is phosphorylated by Mec1p and Tel1p. Gel analysis showed that Cdc13p contains a Mec1/Tel1-dependent posttranslational modification. Using an immunoprecipitate (IP)-kinase assay, we showed that Mec1p phosphorylates Cdc13p on serine 225, 249, 255 and 306, and Tel1p phosphorylates Cdc13p on serine 225, 249, and 255 in vitro. Phenotypic analysis in vivo revealed that the mutations in the Cdc13p SQ motifs phosphorylated by Mec1p and Tel1p caused multiple telomere and growth defects. In addition, normal telomere length and growth could be restored by expressing a Cdc13-Est1p hybrid protein. These results demonstrate the telomerase recruitment domain of Cdc13p as an important new telomere-specific target of Mec1p/Tel1p.
Involvement of DNA polymerases α,β,δ,ε and γ in non-homologous end-joining in Saccharomyces cerevisiae
Most DNA repair pathways require DNA polymerase to execute the step of nucleotide extension. Non-Homologous End-Joining (NHEJ) is required to repair chromosomal double strand breaks and to assemble antigen receptor variable regions in developing B and T lymphocytes. However, the question about which DNA polymerases are involved resolving double strand breaks through NHEJ in vivo is still elusive. NHEJ pathways are observed in the both simple end joining and chromosomal rearrangement. Here we used an in vivo assay to characterize different DNA polymerases and to understand their roles in simple end joining and chromosomal rearrangement in Saccharomyces cerevisiae. All six major DNA polymerases (α,β,γ,δ,ε and ζ) in S. cerevisiae were investigated. Polβ was found to be required in adding bases in simple end joining of NHEJ and it could be physically recruited to double strand break sites. Polε involved in deleting bases through its 3’ to 5’ exonuclease activity in simple end joining of NHEJ. Moreover, both Polδ and Polγ are required in chromosomal rearrangement, but are dispensable for involved simple end joining. Altogether, our results suggest that most DNA polymerases are involved in NHEJ and each plays a distinct role in detail mechanisms of NHEJ.口試委員會審定書 i
誌謝 ii
中文摘要 iii
Abstract v
Contents vii
Chapter 1: Introductions of DNA damage and telomere 1
Chapter 2:The telomerase-recruitment domain of the telomere binding protein Cdc13 is regulated by Mec1p/Tel1p-dependent phosphorylation 4
2.1 Introduction 4
2.2 Materials and methods 7
2.3 Results 12
2.3.1 MEC1/TEL1-dependent Cdc13p phosphorylation in vivo 12
2.3.2 The telomerase recruitment domain of Cdc13p is phosphorylated by Mec1/Tel1 immunoprecipitates 13
2.3.3 S225, S249, S255 and S306 are the in vitro phosphorylation sites of Cdc13p 14
2.3.4 Phenotypic analysis of cdc13-S mutants 15
2.3.5 S249 and S255 of Cdc13p are required for the efficient in vivo action of telomerase 16
2.3.6 Telomere lengthening by targeted Est1p occurs efficiently in the absence of the Mec1/Tel1 phosphorylation sites of Cdc13p 17
2.3.7 Mutations at the Mec1p /Tel1p-phosphorylation sites do not affect the Cdc13p-Pol1p intinteraction 18
2.4 Discussion 19
Chapter 3: Involvement of DNA polymerases α,β,δ,ε and γ in non-homologous end-joining in Saccharomyces cerevisiae 22
3.1 Introduction 22
3.2 Materials and methods 26
3.3 Results 30
3.3.1 Polβ is required for addition of base in simple end-joining of NHEJ 30
3.3.2 Polβ is physically recruited to a DSB 31
3.3.3 Polγ is involved in chromosomal rearrangement 32
3.3.4 Polα is involved in both simple end-joining and chromosomal rearrangement 32
3.3.5 Polδ is involved in chromosomal rearrangement of NHEJ 33
3.3.6 Polε participates in the deletion base pathway in the simple end-joining through its 3’ to 5’ exonuclease activity 33
3.4 Discussion 35
Conclusion 38
Tables and Figures 40
References 66
Appendix: Curriculum Vitae 7
Aclees visayus Meregalli & Boriani & Taddei & Hsu & Tseng & Mouttet 2020
No full textAclees visayus (Heller, 1929) Aclees visayus. Philippines: Mindanao, Surigao del Sur, Barobo, I.2019, 1 ♂, local collector leg.Published as part of Meregalli, Massimo, Boriani, Marco, Taddei, Andrea, Hsu, Chen-Fu, Tseng, Wei-Zhe & Mouttet, Raphaëlle, 2020, A new species of Aclees from Taiwan with notes on other species of the genus (Coleoptera: Curculionidae: Molytinae), pp. 1-26 in Zootaxa 4868 (1) on page 19, DOI: 10.11646/zootaxa.4868.1.1, http://zenodo.org/record/442856
Aclees aenigmaticus Meregalli & Boriani & Taddei & Hsu & Tseng & Mouttet 2020, sp. n.
No full textAclees aenigmaticus sp. n. Aclees aenigmaticus ex. 1. Taiwan: New Taipei City, 5.X.2018, 1 ♂, Chen–Fu Hsu leg. Aclees aenigmaticus ex. 2. Taiwan: New Taipei City, Tamsui District, Tamkang Farm, 5.XI.2017, 1 ♀, Wei-Zhe Tseng leg. Aclees aenigmaticus ex. 3. Taiwan: Taipei City, 11.IX.2016, 1 ♀, Kuang-Ping Yu leg. Aclees aenigmaticus ex. 4. Taiwan: Taipei City, 29.IX.2016, 1 ♀, Chen-Fu Hsu leg. Aclees aenigmaticus ex. 5. Vietnam: Yên Bái, Mù Cang Ch ải, 1.700 m, VIII.2018, 1♂, local collector leg. Aclees aenigmaticus ex. 6. China (Sichuan) : CHINA, Sichuan prov., Mount Qingcheng, 725m, N 30°53.770’ E 103°34.690’, 11-16-v-2017, Walter Grosser leg. Aclees aenigmaticus ex. 7. Taiwan: Nantou County—IX.2016, 1 ♀, local collector leg. Aclees aenigmaticus ex. 8. Taiwan: Taichung City, Heping District, Wuling Farm., 1 ♀, 13.VIII.2017, Sheng-Hung Lin leg. Aclees aenigmaticus ex. 9. Taiwan: New Taipei City, 21.VIII.2015, 1♂, Chen-Fu Hsu leg. Aclees aenigmaticus ex. 10. Taiwan: New Taipei City, Xinyi District, Hushan Hiking Trail, 30.VII.2017, 1 ♀, Wei-Zhe Tseng leg. Aclees aenigmaticus ex. 11. China (Yunnan): W of Yunnan, N of Nujiang valley, Mt. Biluoxueshan, 2100m, 2018.6-7, 1♂, Lina Xu leg. ......continued on the next pagePublished as part of Meregalli, Massimo, Boriani, Marco, Taddei, Andrea, Hsu, Chen-Fu, Tseng, Wei-Zhe & Mouttet, Raphaëlle, 2020, A new species of Aclees from Taiwan with notes on other species of the genus (Coleoptera: Curculionidae: Molytinae), pp. 1-26 in Zootaxa 4868 (1) on pages 20-21, DOI: 10.11646/zootaxa.4868.1.1, http://zenodo.org/record/442856
Identification and Mapping of the QTL Controlling Resistance to Blast (Magnaporthe grisea) Disease in Rice
No full textOmophorus (Sinomophorus) wallaCei: a new weevil from Borneo highlights the enigmatic Ethiopian-Oriental disjunct distribution (Coleoptera, Curculionidae, Molytinae)
No full textTseng, Wei-Zhe, Hsiao, Yun, Hsu, Chen-Fu (2018): Omophorus (Sinomophorus) wallaCei: a new weevil from Borneo highlights the enigmatic Ethiopian-Oriental disjunct distribution (Coleoptera, Curculionidae, Molytinae). Zootaxa 4438 (3): 588-596, DOI: 10.11646/zootaxa.4438.3.1
Proofreading Activity of DNA Polymerase Pol2 Mediates 3'-End Processing during Nonhomologous End Joining in Yeast
No full textGenotoxic agents that cause double-strand breaks (DSBs) often generate damage at the break termini. Processing enzymes, including nucleases and polymerases, must remove damaged bases and/or add new bases before completion of repair. Artemis is a nuclease involved in mammalian nonhomologous end joining (NHEJ), but in Saccharomyces cerevisiae the nucleases and polymerases involved in NHEJ pathways are poorly understood. Only Pol4 has been shown to fill the gap that may form by imprecise pairing of overhanging 39 DNA ends. We previously developed a chromosomal DSB assay in yeast to study factors involved in NHEJ. Here, we use this system to examine DNA polymerases required for NHEJ in yeast. We demonstrate that Pol2 is another major DNA polymerase involved in imprecise end joining. Pol1 modulates both imprecise end joining and more complex chromosomal rearrangements, and Pol3 is primarily involved in NHEJ -mediated chromosomal rearrangements. While Pol4 is the major polymerase to fill the gap that may form by imprecise pairing of overhanging 3' DNA ends, Pol2 is important for the recession of 39 flaps that can form during imprecise pairing. Indeed, a mutation in the 3'- 5' exonuclease domain of Pol2 dramatically reduces the frequency of end joins formed with initial 3' flaps. Thus, Pol2 performs a key 3' end-processing step in NHEJ
Aclees visayus Meregalli & Boriani & Taddei & Hsu & Tseng & Mouttet 2020, comb. n.
No full textAclees visayus (Heller, 1929) comb. n. Fig. 9 Pagiophloeus visayus Heller, 1929: 12. Type locality: Philippine Islands, Samar. Heller (1929) described 7 species of Molytinae from the Philippine Islands as Pagiophloeus Faust, 1892 (a synonym of Pimelocerus Lacordaire, 1863), including P. visayus. One of the authors (M.M.) examined the type specimen of P. visayus, conserved at the MTD. Based on its morphology, it is transferred to the genus Aclees as Aclees visayus (Heller, 1929), comb. n. In the molecular analysis a specimen morphologically corresponding to the type of P. visayus was part of the Aclees clade, and it clustered as sister to the other species examined.Published as part of Meregalli, Massimo, Boriani, Marco, Taddei, Andrea, Hsu, Chen-Fu, Tseng, Wei-Zhe & Mouttet, Raphaëlle, 2020, A new species of Aclees from Taiwan with notes on other species of the genus (Coleoptera: Curculionidae: Molytinae), pp. 1-26 in Zootaxa 4868 (1) on pages 16-17, DOI: 10.11646/zootaxa.4868.1.1, http://zenodo.org/record/442856
A low-cost neural sorting network with O(1) time complexity
Full text link[[abstract]]In this paper, we present an O(1) time neural network with O(n1 + var epsilon) neurons and links to sort n data, var epsilon > 0. For large-size problems, it is desirable to have low-cost hardware solutions. In order to solve the sorting problem in constant time and with less hardware-cost, we adopt Leighton's column sort [5] as the main architecture. Then we use Chen and Hsieh's neural network [3] with O(n3) complexity as the lowest-level sub-networks. By using recursive techniques properly, we are able to explore constant-time, low-complexity neural sorting networks.
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