Error Prone vs Error Free DNA Replication in Human Cells

人类细胞中容易出错的 DNA 复制与无错误的 DNA 复制

• 批准号: 6794173
• 负责人: VERONICA M MAHER
• 金额: $ 34.76万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6794173
• 关键词: DNA damage; DNA directed DNA polymerase; DNA repair; DNA replication; complementary DNA; enzyme activity; fibroblasts; gene conversion; genetic recombination; human tissue; hypoxanthine phosphoribosyltransferase; nucleic acid sequence; polymerase chain reaction; tissue /cell culture

The overall goals of the proposed studies are: to examine in human cells the mechanisms involved in "translesion synthesis"(TLS), i.e., the process by which human cells insert nucleotides opposite lesions in the DNA template that block replication by the major replication complex; to examine the mechanisms involved in "damage avoidance", a broad term that refers to various error-free processes by which human cells complete replication without using the damaged DNA as a template; and to determine whether there is a reciprocal relationship between these two alternative pathways. 1.) We will test the hypothesis that when replication by the normal replication complex is blocked by lesions, TLS, using the damaged DNA as a template, involves the products of several specialized human polymerases and the chance that a specific polymerase will introduce "spontaneous" or mutagen-induced mutations during TLS depends upon: a) the nature of the lesion; b) whether it is located in the template strand for leading or lagging strand synthesis; c) the surrounding sequence; and d) the availability of the other competing specialized polymerases in the cell; 2.) We will test the hypothesis that when DNA replication is blocked, human fibroblasts obtain the necessary genetic information from an undamaged homologous copy of the DNA, e.g., the newly- replicated daughter-strand of the corresponding DNA, i.e., the sister chromatid or the corresponding allele (recombination, gene conversion); 3.) We will test the hypothesis that if such "damage avoidance" pathways for obtaining the genetic information from homologous copies of DNA are blocked, use of mutagenic TLS will increase correspondingly, i.e., the relationship is reciprocal; 4.) We will determine a) the nature of the "damage-avoidance" mechanism(s) used to complete lesion-blocked DNA replication b) the relative frequency at which cells use those pathways rather than TLS pathways, and c) the effect of lack of particular gene products on the (reciprocal) relationship between the pathways, using substrates designed to reveal the mechanisms used, from the nature of the products.

拟议研究的总体目标是：在人类细胞中检查“跨损伤合成”(TLS)所涉及的机制，即人类细胞在DNA模板中插入与损伤相对的核苷酸以阻止主要复制复合体复制的过程；检查“损伤避免”所涉及的机制；“损伤避免”是一个宽泛的术语，指的是人类细胞在不使用受损DNA作为模板的情况下完成复制的各种无错误过程；并确定这两种替代途径之间是否存在相互作用的关系。1)我们将测试这样一种假设，即当正常复制复合体的复制被病变阻止时，使用受损的DNA作为模板的TLS涉及几种专业人类聚合酶的产物，以及特定的聚合酶在TLS过程中引入“自发”或诱变剂诱导突变的机会取决于：a)病变的性质；b)它是否位于模板链中，用于领先或滞后链合成；c)周围序列；以及d)细胞中其他竞争的专业聚合酶的可用性；2)我们将测试这样一种假设，即当DNA复制被阻止时，人类成纤维细胞从未受损的DNA同源拷贝中获得必要的遗传信息，例如，相应DNA的新复制的子链，即姐妹染色单体或相应的等位基因(重组、基因转换)；我们将检验这样一种假设，即如果从DNA的同源拷贝中获得遗传信息的这种“损害避免”途径被阻止，突变TLS的使用将相应增加，即这种关系是互惠的；我们将确定a)用于完成损伤阻断的dna复制的“损伤-避免”机制(S)的性质，b)细胞使用这些途径而不是TLS途径的相对频率，以及c)缺乏特定的基因产物对途径之间(相互)关系的影响，使用的底物旨在揭示所使用的机制，从产品的性质出发。