REGULATION OF THE BREAST CELL DNA SYNTHESOME

乳腺细胞 DNA 合成体的调节

• 批准号: 2372121
• 负责人: ROBERT J HICKEY
• 金额: $ 18.61万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-18 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2372121
• 关键词: ADP ribosylation; DNA directed DNA polymerase; DNA replication; SDS polyacrylamide gel electrophoresis; adenine phosphoribosyltransferase; breast neoplasms; cell line; chemical kinetics; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; gel mobility shift assay; mammary gland; molecular weight; nucleic acid biosynthesis

DESCRIPTION: The genetic damage which accompanies the development and progression of breast cancer has been linked to defects in the DNA synthetic and DNA repair processes of these cells; and these defects correlate with changes in the physical structure and enzymatic activity of several proteins used to carry-out DNA synthesis and repair. The investigator's data indicate that, as compared to normal breast cells, breast cancer cells exhibit a 3-4-fold higher DNA synthetic rate, a 6-8-fold decrease in replication fidelity, and have specific structural alterations in several of the proteins used to carry-out DNA replication. His goal, therefore, is to establish a clear link between the differences in the DNA synthetic activity and replication fidelity of normal and malignant breast cells and the structural changes specific replication essential proteins undergo during transformation. To accomplish this, he will initially focus on defining the kinetic, biophysical, and detailed structural properties of the DNA polymerases and poly(ADP-ribose) polymerase (PARP) by determining: 1) differences in the kinetic properties of these enzymes between the normal and malignant breast cells looking specifically at the Km and Vmax for substrate utilization, substrate specificity, and the proof-reading activity of the intrinsic 3-5' exonuclease of DNA polymerases delta and epsilon; as well as the fidelity with which all of the DNA polymerases carry-out DNA synthesis; 2) differences in the biophysical characteristics of normal and malignant breast cell DNA polymerases alpha, delta, epsilon and PARP looking specifically at the molecular weight, Stokes radii, sedimentation coefficient, isoelectric point, and 2D-PAGE profiles of these enzymes; and 3) differences in the 2D-PAGE tryptic peptide maps of the normal and malignant breast cell polymerases looking specifically at the role of phosphorylation, poly(ADP-ribosylation), and primary amino acid sequence in mediating these differences.

描述：伴随着发育和 乳腺癌的进展与DNA合成缺陷有关 以及这些细胞的DNA修复过程;这些缺陷与 几种蛋白质的物理结构和酶活性的变化 用来进行DNA合成和修复。 研究者的数据 表明与正常乳腺细胞相比，乳腺癌细胞 表现出3-4倍的高DNA合成速率，6-8倍的降低， 复制保真度，并且在几个中具有特定的结构改变 用于进行DNA复制的蛋白质。 因此，他的目标是 建立DNA合成活性差异之间的明确联系 正常和恶性乳腺细胞的复制保真度， 复制过程中特定复制必需蛋白质的结构变化 转型 为了实现这一目标，他将首先专注于定义 DNA的动力学、生物物理学和详细结构特性 聚合酶和聚（ADP-核糖）聚合酶（PARP），通过测定：1） 这些酶的动力学性质的差异之间的正常 而恶性乳腺癌细胞则特别关注Km和Vmax， 底物利用率、底物特异性和校对活性 DNA聚合酶delta和delta的固有3-5'外切核酸酶; 以及所有DNA聚合酶携带DNA的保真度 2）正常和异常的生物物理特征的差异， 恶性乳腺细胞DNA聚合酶α、δ、β和PARP 特别是在分子量，斯托克斯半径，沉降 这些酶的系数、等电点和2D-PAGE图谱;以及 3)正常人和正常人的2D-PAGE胰蛋白酶肽图谱的差异 恶性乳腺细胞聚合酶特别关注 磷酸化，聚（ADP-核糖基化），和一级氨基酸序列， 调解这些差异。