Telomere terminal extension and replication: mechanisms and links to DNA repair

端粒末端延伸和复制：DNA 修复的机制和联系

• 批准号: 10809126
• 负责人: NEAL F LUE
• 金额: $ 1.72万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10809126
• 关键词: BRCA2 gene; Binding; Biochemical; Biochemistry; Biological Assay; Bone marrow failure; Candida glabrata; Chromatin; Chromosomes; Complex; DNA; DNA Primase; DNA Repair; DNA biosynthesis; Defect; Development; Disease; Exhibits; Genetic; Genetic Recombination; Genome; Genome Stability; Investigation; Link; Liver Fibrosis; Lung; Malignant Neoplasms; Mammals; Modeling; Molecular Conformation; Nucleoproteins; Pathway interactions; Proteins; RNA-Directed DNA Polymerase; Regulation; Research; Resolution; Role; Structure; Telomerase; Telomere Capping; Ustilago; Work; biological adaptation to stress; fungus; genome-wide; helicase; insight; novel; novel diagnostics; novel therapeutics; prevent; repaired; replication stress; single-molecule FRET; telomere

Project Summary/Abstract Telomeres, the specialized nucleoprotein structures located at the ends of eukaryotic chromosomes, are critical for genome stability. Telomere DNA, which consists of numerous copies of a short repeat, is difficult to maintain owing to (1) the end replication problem that prevents the complete duplication of parental DNA; and (2) the propensity of telomere DNA and chromatin to form replication barriers. The main players that help to overcome these difficulties include (1) telomerase, a special reverse transcriptase that adds “G-strand” repeats onto the 3’ ends of chromosomes; (2) primase-Pol a (PP), which adds “C-strand” repeats onto the 5’ ends of chromosomes; and (3) helicases and repair proteins that facilitate semi-conservative replication through telomeres. Telomerase has been subjected to detailed investigation and much is known about its mechanisms and regulation. Hence, in this application, we will focus on the roles of primase-Pol a and repair proteins such as Rad51 and Brh2 (BRCA2). The study will employ two fungal models (Candida glabrata and Ustilago maydis), each with its own unique advantages. In Aim 1, we will examine the mechanisms of PP and its regulation by CST, a telomere binding complex. We have identified a critical and conserved interface between the Stn1 and Pol12 subunits of CST and PP, and shown that this interaction likely triggers a conformational switch in PP to facilitate DNA synthesis. We will address this novel conformational switch mechanism using a combination of biochemistry, cyroEM and smFRET. In addition, both CST and PP have been linked to telomere replication and genome-wide replication stress response, though the underlying mechanisms are poorly understood. Accordingly, we will dissect the role of the CST-PP interaction in these pathways. These studies will be conducted using C. glabrata proteins because they are easily purified and biochemically tractable. In Aim 2 – 3, we will address the mechanisms of two core repair proteins (Rad51 and Brh2[BRCA2]) in telomere replication and telomere capping. we have developed a high- resolution assay for telomere replication defects and used the assay to demonstrate critical functions for several repair proteins. We have also uncovered a novel and conserved interaction between Rad51 the telomere protein Pot1, which suggests novel, telomere-specific regulatory mechanisms. Hence in these two aims, we will dissect the mechanisms of Rad51 at telomeres and determine how its functions are regulated by Pot1 and Brh2 using a combination of genetics and biochemistry. Because RAD51 and BRCA2 factors have also been implicated in promoting replication and stabilizing stalled forks throughout the genome, our work may lead to a more integrated view of their mechanisms. This investigation will be carried out using Ustilago maydis because unlike standard fungi, U. maydis exhibits a high degree of similarity to mammals with respect to the recombination and telomere machinery. 8

项目总结/文摘

项目成果

STN1-POLA2 interaction provides a basis for primase-pol α stimulation by human STN1.

• DOI: 10.1093/nar/gkx621
• 发表时间: 2017-09-19
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Ganduri S;Lue NF
• 通讯作者: Lue NF

Evolving Linear Chromosomes and Telomeres: A C-Strand-Centric View.

• DOI: 10.1016/j.tibs.2018.02.008
• 发表时间: 2018-05
• 期刊: Trends in biochemical sciences
• 影响因子: 13.8
• 作者: Lue NF

Telomere recombination pathways: tales of several unhappy marriages.

• DOI: 10.1007/s00294-016-0653-8
• 发表时间: 2017-06
• 期刊: Current genetics
• 影响因子: 2.5
• 作者: Lue NF;Yu EY
• 通讯作者: Yu EY

The mechanisms of K. lactis Cdc13 in telomere DNA-binding and telomerase regulation.

乳酸克鲁维酵母 Cdc13 在端粒 DNA 结合和端粒酶调节中的机制。

• DOI: 10.1016/j.dnarep.2017.11.007
• 发表时间: 2018
• 期刊: DNA repair
• 影响因子: 3.8
• 作者: Hsu,Min;Lue,NealF
• 通讯作者: Lue,NealF

Molecular architecture and oligomerization of Candida glabrata Cdc13 underpin its telomeric DNA-binding and unfolding activity.

念珠菌CDC13的分子结构和寡聚的基础是其端粒DNA结合和展开活性。

• DOI: 10.1093/nar/gkac1261
• 发表时间: 2023-01-25
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Coloma, Javier;Gonzalez-Rodriguez, Nayim;Balaguer, Francisco A.;Gmurczyk, Karolina;Aicart-Ramos, Clara;Nuero, oscar M.;Luque-Ortega, Juan Roman;Calugaru, Kimberly;Lue, Neal F.;Moreno-Herrero, Fernando;Llorca, Oscar
• 通讯作者: Llorca, Oscar