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（申请人提供）：阿糖胞苷（β-D-阿拉伯呋喃糖基胞嘧啶，araC）用于治疗急性髓性白血病（AML）已有 40 多年的历史，AraCTP 与 dCTP 竞争掺入 DNA，从而发挥化疗作用。 araC 的作用源自其抑制 DNA 复制的能力，尽管复制聚合酶 (Pols) 无法插入。由于 araCTP 位于新合成 DNA 的 3' 末端，因此它们的延伸受到抑制。然而，人类细胞具有许多跨损伤合成 (TLS) DNA Pol，原则上可以克服 araC 对 DNA 复制的抑制作用。既从 araC 终止的 3' 末端延伸 DNA 合成，又通过掺入模板链的 araC 损伤进行复制，以了解 TLS Pols 在促进 DNA 复制中的相对重要性和机制。 araC 损伤的 DNA，我们将结合遗传、细胞、生物化学和结构研究在目标 1 中，我们将 (a) 分析 TLS Pols 在通过 araC 在人类细胞中介导复制中的作用，并确定它们是否起作用。以无错误或诱变的方式检查 araC 损伤的 DNA 复制所需的 TLS Pols 的消耗对 araC 处理的人类细胞中复制叉的进展的影响； (c) 检查 TLS Pols 的消耗对 araC 处理的人类细胞存活的影响 在目标 2 中，我们将进行稳态动力学分析，以 (a) 确定 TLS Pols 在 araC 延伸时的催化效率。 3' 引物末端；(b) 确定 TLS Pols 对 araC 掺入核苷酸 (nt) 的熟练程度和保真度；以及 (c) 分析熟练程度此外，（d）我们将进行预稳态动力学研究以获得更好的结果 了解 TLS Pols 从 araC 延伸以及在 araC 对面插入 nts 的机制 在目标 3 中，我们将确定 TLS Pols 的三元复杂晶体结构，其 (a) 在模板链中插入 araC 对面的 nts；在 TLS 的延伸步骤中，当 araC 与引物末端配对时插入正确或不正确的 nt；以及 (c) DNA 合成延伸所需的总之，这些研究将更深入地了解不同的 TLS Pols 在促进 araC 受损 DNA 复制中所起的作用，以及 TLS Pols 如何设法将 araC 容纳到其活性位点及其作用动力学机制。除了关于 TLS Pols 在 araC 受损 DNA 复制中的作用的新机制信息之外，这些研究还可能提出提高 araC 化疗治疗复发为主要问题的癌症有效性的方法。