Genetic and molecular mechanisms of replication of araC damaged DNA

araC 损伤 DNA 复制的遗传和分子机制

• 批准号: 9188059
• 负责人: SATYA PRAKASH
• 金额: $ 40.34万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9188059
• 关键词: Active Sites; Acute Myelocytic Leukemia; Biochemical; Cancer Relapse; Cells; Complex; Crystallization; Cytarabine; Cytosine; DNA; DNA Damage; DNA biosynthesis; DNA replication fork; Data; Disease remission; Effectiveness; Genetic; Human; Immunotherapy; Income; Kinetics; Lesion; Light; Mediating; Molecular; Mus; Nucleotides; Patients; Pharmaceutical Preparations; Plasmids; Play; Polymerase; Relapse; Replication Origin; Role; Simian virus 40; Site; Stem cell transplant; Structure; System; base; chemotherapy; cytotoxic; novel; nucleoside analog; public health relevance; treatment choice; tripolyphosphate

 DESCRIPTION (provided by applicant): Cytarabine (β-D-arabinofuranosyl cytosine, araC) has been used for the treatment of acute myelogenous leukemia (AML) for over 40 years. AraCTP competes with dCTP for incorporation into DNA; thus, the chemotherapeutic action of araC derives from its ability to inhibit DNA replication. Although the replicative polymerases (Pols) ca insert araCTP at the 3' terminus of newly synthesized DNA, they are inhibited at extending from it. However, human cells harbor a number of translesion synthesis (TLS) DNA Pols that can, in principle, overcome the inhibitory effects of araC on DNA replication by both extending DNA synthesis from araC-terminated 3' ends and by replicating through the araC lesion that becomes incorporated into the template strand. To understand the relative significance and mechanisms of TLS Pols in promoting replication of araC-damaged DNA, we will carry out a combination of genetic, cellular, biochemical, and structural studies. In Aim 1, we will (a) analyze the roles of TLS Pols in mediating replication through araC in human cells and determine whether they act in an error-free or mutagenic manner; (b) examine the effects of depletions of TLS Pols required for the replication of araC-damaged DNA on the progression of the replication fork in araC treated human cells; and (c) examine the effects of depletions of TLS Pols on the survival of human cells treated with araC. In Aim 2, we will carry out steady- state kinetic analyses to (a) determine the catalytic efficiencies of TLS Pols for extending from araC at the 3' primer terminus; (b) determine the proficiency and fidelity of TLS Pols for nucleotide (nt) incorporation opposite araC; and (c) analyze the proficiency of TLS Pols for extending from the nt inserted opposite araC; in addition, (d) we will carry out pre-steady state kinetic studies to gain a better understanding of the mechanisms of TLS Pols in extending from araC and in inserting nts opposite araC. In Aim 3, we will determine ternary complex crystal structures of TLS Pols that (a) insert nts opposite araC in the templating strand; (b) that function in the extension step of TLS by inserting the correct or incorrect nt when araC is paired to the primer terminus; and (c) that are required for the extension of DNA synthesis from araC terminated DNA. Altogether, these studies will provide a deeper understanding of the roles that different TLS Pols play in promoting the replication of araC damaged DNA, and how the TLS Pols manage to accommodate araC into their active sites and their kinetic mechanisms of action. In addition to novel mechanistic information on the roles of TLS Pols in the replication of araC damaged DNA, these studies may posit ways to increase the effectiveness of araC chemotherapy for the treatment of a cancer where relapse is a major problem.

 描述（由应用提供）：细胞链滨（β-D-阿拉伯呋喃糖基胞嘧啶，ARAC）已用于治疗40多年的急性髓质白血病（AML）。 Aractp与DCTP竞争DNA的保险；因此，ARAC的化学治疗作用来自其抑制DNA复制的能力。尽管复制性聚合酶（POLS）Ca在新合成的DNA的3'末端插入了ARACTP，但它们在从中延伸时受到抑制。然而，人类细胞具有许多转移合成（TLS）DNA pol，这些DNA原则上可以通过从ARAC端接的3'末端以及通过ARAC病变复制而通过ARAC终止的DNA合成来克服ARAC对DNA复制的抑制作用，从而使ARAC终止的抑制作用变成模板链中。为了了解TLS POLS在促进ARAC受损DNA复制中的相对意义和机制，我们将进行遗传，细胞，生化和结构研究的组合。在AIM 1中，我们将（a）分析TLS POLS在人类细胞中通过ARAC复制中介导复制的作用，并确定它们是否以无错误或诱变的方式起作用； （b）检查复制ARAC受损DNA所需的TLS POLS部署的影响，对ARAC处理过的人类细胞中复制叉的进展； （c）检查TLS POLS部署对用ARAC处理的人类细胞存活的影响。在AIM 2中，我们将进行稳态的动力学分析，以确定TLS POL的催化效率，用于从ARAC延伸到3'Prime末端； （b）确定TLS POLS对核苷酸（NT）的熟练程度和保真度； （c）分析TLS pol的熟练度，以从插入的NT延伸到对面的ARAC相对；此外，（d）我们将进行稳态的状态动力学研究，以获得更好的 了解从ARAC延伸和插入ARAC对面的NTS中TLS POL的机制。在AIM 3中，我们将确定（a）在模板链中插入ARAC的TLS pol的三元复合物晶体结构； （b）当将ARAC配对与引物末端配对时，通过插入正确或错误的NT来在TLS的扩展步骤中发挥作用； （c）从ARAC终止DNA扩展DNA合成所需的。总的来说，这些研究将提供对不同TLS POLS在促进ARAC损坏的DNA复制中发挥作用的作用的更深入的理解，以及TLS POLS如何设法将ARAC容纳到其活性位点及其动力学机制。除了有关TLS POLS在ARAC损坏DNA复制中的作用的新型机械信息外，这些研究还可以提高ARAC化学疗法在治疗癌症治疗的有效性的积极方法，而缓解是主要问题。