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: 9000855
• 负责人: SATYA PRAKASH
• 金额: $ 41.72万
• 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/9000855
• 关键词: Active Sites; Acute Myelocytic Leukemia; Biochemical; Cancer Relapse; Cells; Complex; Cytarabine; Cytosine; DNA; DNA Damage; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Data; Disease remission; Effectiveness; Genetic; Human; Immunotherapy; Kinetics; Lesion; Light; Mediating; Molecular; Molecular Genetics; Mus; Nucleotides; 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; relapse patients; 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（由申请方提供）：阿糖胞苷（β-D-阿拉伯呋喃糖基胞嘧啶，araC）已用于治疗急性髓性白血病（AML）超过40年。AraCTP与dCTP竞争掺入DNA;因此，araC的化疗作用源自其抑制DNA复制的能力。尽管复制型聚合酶（Pos）可以在新合成的DNA的3 '末端插入araCTP，但是它们在从其延伸时受到抑制。然而，人类细胞具有许多跨损伤合成（TLS）DNA Pos，原则上，通过延长araC的DNA合成，克服araC对DNA复制的抑制作用，终止的3 '末端，并通过掺入模板链的araC损伤进行复制。为了了解TLS Pos在促进araC损伤DNA复制中的相对意义和机制，我们将进行遗传，细胞，生物化学和结构研究的组合。在目的1中，我们将（a）分析TLS Pos在人类细胞中通过araC介导复制中的作用，并确定它们是否以无错误或致突变的方式起作用;（B）检查araC损伤的DNA复制所需的TLS Pos的消耗对araC处理的人类细胞中复制叉进展的影响;和（c）检查TLS Pol的耗尽对用araC处理的人细胞的存活的影响。在目的2中，我们将进行稳态动力学分析以（a）确定TLS Pos从3 '引物末端的araC延伸的催化效率;（B）确定TLS Pos相对于araC掺入核苷酸（nt）的能力和保真度;和（c）分析TLS Pos从插入的nt相对于araC延伸的能力;此外，（四）我们会进行稳定前的动力学研究， 理解TLS Pol从araC延伸和在araC对面插入nts的机制。在目标3中，我们将确定TLS Pol的三元复合物晶体结构，其（a）在模板链中相对于araC插入nt;（B）当araC与引物末端配对时，通过插入正确或不正确的nt在TLS的延伸步骤中起作用;以及（c）从araC终止的DNA延伸DNA合成所需的。 总之，这些研究将提供对不同TLS Pos在促进araC损伤DNA复制中所起作用的更深入了解，以及TLS Pos如何设法将araC容纳到其活性位点及其动力学作用机制。除了关于TLS Pos在araC损伤DNA复制中的作用的新机制信息之外，这些研究可能会增加araC化疗治疗复发是主要问题的癌症的有效性。