DNA Replication, Repair, and Mutagenesis In Eukaryotic And Prokaryotic Cells

真核和原核细胞中的 DNA 复制、修复和诱变

基本信息

批准号：
9550317
负责人：
ROGER WOODGATE
金额：
$ 207.5万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9550317
关键词：
129X1/SvJ Mouse Amino Acids Antibodies Archaea Bacteria Baculoviridae Baculoviruses Biochemical Biological Process Catalysis Cells Characteristics Codon Nucleotides Complementary DNA DNA DNA Damage DNA biosynthesis DNA lesion DNA polymerase A DNA polymerase iota DNA sequencing DNA-Directed DNA Polymerase Enzymes Eukaryota Eukaryotic Cell Exhibits Exons Family Fishes Genes Genomics Glutathione Human Immunoprecipitation In Vitro Initiator Codon Insecta Ions Laboratories Lead Length Magnesium Manganese Messenger RNA Metals Molecular Mouse Strains Mutagenesis Mutation N-terminal Nonsense Mutation Organism Pathway interactions Peptides Polymerase Process Prokaryotic Cells Property Protein Isoforms Proteins Reaction Recombinants Reporting Scientist Technology Testis Time Western Blotting Wild Type Mouse base genome integrity human DNA in vitro activity in vivo molecular sequence database pol Gene Products repaired

项目摘要

Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into DNA. These studies have traditionally spanned the evolutionary spectrum and include studies in bacteria, archaea and eukaryotes. In the past year, our studies have focused on human DNA polymerase iota: The cDNA encoding human DNA polymerase iota (POLI) was cloned in 1999. At that time, it was believed that the POLI gene encoded a protein of 715 amino acids. Advances in DNA sequencing technologies lead to the realization that there is an upstream, in-frame initiation codon that would encode a DNA polymerase iota (pol iota) protein of 740 amino acids. The extra 25 amino acid region is rich in acidic residues (11/25) and is reasonably conserved in eukaryotes ranging from fish to humans. As a consequence, the curated Reference Sequence (RefSeq) database identified pol-iota as a 740 amino acid protein. However, the existence of the 740 amino acid pol iota protein has never been shown experimentally. Using highly specific antibodies to the 25 N-terminal amino acids of pol iota, we were unable to detect the longer 740 amino acid (iota-long) isoform in western blots. However, trace amounts of the iota-long isoform were detected after enrichment by immunoprecipitation. One might argue that the longer isoform may have a distinct biological function, if it exhibits significant differences in its enzymatic properties from the shorter, well-characterized 715 amino acid pol iota. We therefore purified and characterized recombinant full-length (740 amino acid) pol iota-long and compared it to full-length (715 amino acid) pol iota-short in vitro. The metal ion requirements for optimal catalytic activity differ slightly between pol iota-long and pol iota-short, but under optimal conditions, both isoforms exhibit indistinguishable enzymatic properties in vitro. We also report that like pol iota-short, the pol iota-long isoform can be monoubiquitinated and polyubiuquitinated in vivo, as well as form damage induced foci in vivo. We conclude that the predominant isoform of pol iota in human cells is the shorter 715 amino acid protein and that if, or when, expressed, the longer 740 amino acid isoform has identical properties to the considerably more abundant shorter isoform. In 2003, we reported that 129-derived strains of mice carry a naturally occurring nonsense mutation at codon 27 of the Poli gene that would produce a pol iota peptide of just 26 amino acids, rather then the full-length 717 amino acid wild-type polymerase. In support of the genomic analysis, no pol iota protein was detected in testes extracts from 129X1/SvJ mice, where wild-type pol iota is normally highly expressed. The early truncation in pol iota occurs before any structural domains of the polymerase are synthesized and as a consequence, we reasoned that 129-derived strains of mice should be considered as functionally defective in pol iota activity. However, it has recently been reported that during the maturation of the Poli mRNA in 129-derived strains, exon- 2 is sometimes skipped and that an exon-2-less pol protein of 675 amino acids is synthesized that retains catalytic activity in vitro and in vivo. From a structural perspective, we found this idea untenable, given that the amino acids encoded by exon-2 include residues critical for the coordination of the metal ions required for catalysis, as well as the structural integrity of the DNA polymerase. To determine if the exon-2-less pol iota isoform possesses catalytic activity in vitro, we purified a glutathione-tagged full-length exon-2-less (675 amino acid) pol iota protein from baculovirus infected insect cells and compared the activity of the isoform to full-length (717 amino acid) GST-tagged wild-type mouse pol iota in vitro. Reaction conditions were performed under a range of magnesium or manganese concentrations, as well as different template sequence contexts. Wild-type mouse pol iota exhibited robust characteristic properties previously associated with human pol iotas biochemical properties. However, we did not detect any polymerase activity associated with the exon-2-less pol iota enzyme under the same reaction conditions and conclude that exon-2-less pol iota protein is indeed rendered catalytically inactive in vitro.

基因组完整性实验室（LGI）中的科学家研究了将突变引入DNA的机制。这些研究传统上跨越了进化谱，包括细菌，古细菌和真核生物的研究。在过去的一年中，我们的研究集中在人类DNA聚合酶IOTA上：编码人DNA聚合酶IOTA（poli）的cDNA于1999年克隆。当时，人们认为poli基因编码了715个氨基酸的蛋白质。 DNA测序技术的进步导致人们意识到有一个上游，框架内的密码子可以编码740个氨基酸的DNA聚合酶IOTA（POL IOTA）蛋白。额外的25个氨基酸区域富含酸性残基（11/25），并且在从鱼到人类的真核生物中得到了合理的保守。结果，策划的参考序列（REFSEQ）数据库将POL-iota鉴定为740个氨基酸蛋白。但是，从未通过实验表现出740个氨基酸pol Iota蛋白的存在。使用对POL IOTA的25个N末端氨基酸的高度特异性抗体，我们无法检测到蛋白质印迹中更长的740氨基酸（IOTA-long）同工型。但是，通过免疫沉淀富集后检测到痕量的IOTA长同工型。有人可能会争辩说，如果同工型与较短的，较短的715氨基酸pol iota具有显着差异，则可能具有不同的生物学功能。因此，我们纯化并表征了重组全长（740氨基酸）pol iota-long，并将其与全长（715个氨基酸）pol iota-short进行了比较。最佳催化活性的金属离子要求在Pol Iota-long和Pol Iota-short之间略有不同，但是在最佳条件下，两种同工型在体外表现出无法区分的酶促性能。我们还报告说，像Pol Iota-Short一样，Pol Iota-long同工型可以在体内进行单液化和多氨基化，并形成损害诱导的灶在体内。我们得出的结论是，人类细胞中POL IOTA的主要同工型是较短的715氨基酸蛋白，如果或何时表达，则较长的740氨基酸同工型具有相同的特性，与相当大的短相结合型。在2003年，我们报道了129个衍生的小鼠菌株在poli基因的密码子27上携带一种天然存在的胡说八道突变，该突变将产生仅26个氨基酸的poli iota肽，而不是全长的717氨基酸野生型野生型聚合酶。为了支持基因组分析，在129x1/svj小鼠的睾丸提取物中未检测到POL IOTA蛋白，其中通常高度表达野生型POL IOTA。 POL IOTA中的早期截断发生在合成聚合酶的任何结构结构域之前，因此，我们认为应将129个衍生的小鼠菌株视为POL IOTA活性在功能上有缺陷。然而，最近据报道，在129个衍生的菌株中poli mRNA成熟期间，有时会跳过外显子2，并且合成了675个氨基酸的无外显子蛋白，从而在体外和体内保留催化活性。从结构的角度来看，我们发现这一想法是站不住脚的，因为外观2编码的氨基酸包括对催化所需的金属离子以及DNA聚合酶的结构完整性至关重要的残基。为了确定在体外的无外显子体IOTA同工型是否具有催化活性，我们纯化了来自杆状病毒感染昆虫细胞的谷胱甘肽标记的全长外显子2-无（675个氨基酸）POL IOTA蛋白，并比较了同工型的活性，并将其与全长（717氨基酸）的Gerty-prol-prol-prol-prol-betrouty进行了比较。反应条件是在一系列镁或锰浓度以及不同模板序列环境下进行的。野生型小鼠POL IOTA具有以前与人类iotas生化特性相关的鲁棒特性。但是，我们没有在相同的反应条件下检测到与Exon-2无外Eota酶相关的任何聚合酶活性，并得出结论，实际上确实在体外催化了催化性非活性。