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

基因组完整性实验室(LGI)的科学家研究将突变引入DNA的机制。这些研究传统上跨越了进化的光谱，包括对细菌、古菌和真核生物的研究。 在过去的一年里，我们的研究主要集中在人类DNA聚合酶基因座上： 编码人DNA聚合酶IOTA(Poli)的基因于1999年被克隆。当时，人们认为Poli基因编码一种715个氨基酸的蛋白质。DNA测序技术的进步导致人们意识到，存在一个上游的框内起始密码子，它将编码一个由740个氨基酸组成的DNA聚合酶IOTA(Poliota)蛋白。额外的25个氨基酸区域富含酸性残基(11/25)，在从鱼类到人类的真核生物中相当保守。因此，精选参考序列(RefSeq)数据库将Poliota鉴定为740个氨基酸的蛋白质。然而，从实验上从未证明过740个氨基酸残基蛋白的存在。使用针对POLIOTA的25个N-末端氨基酸的高度特异性抗体，我们不能在蛋白质印迹中检测到较长的740个氨基酸(IOTA-Long)亚型。然而，通过免疫沉淀富集后，检测到微量的IOTA-Long异构体。有人可能会争辩说，如果较长的异构体在酶性质上与较短的、特征明确的715氨基酸多糖体有显著差异，那么它可能具有不同的生物学功能。因此，我们纯化并鉴定了重组全长(740个氨基酸)PolIOTA-Long，并在体外将其与全长(715个氨基酸)Poliota-Short进行了比较。Poliota-long和Poliota-Short对最佳催化活性的金属离子要求略有不同，但在最佳条件下，这两种异构体在体外表现出难以区分的酶性质。我们还报道，与Poliota-Short一样，Poliota-Long亚型在体内可以是单泛素和多泛素的，也可以在体内形成损伤诱导的灶。我们的结论是，在人类细胞中，Poliota的主要亚型是较短的715氨基酸蛋白质，如果表达，或当表达时，较长的740氨基酸亚型与相当丰富的较短的亚型具有相同的性质。 2003年，我们报道了129个来源的小鼠在poli基因第27密码子上自然产生的无义突变，该突变将产生一个只有26个氨基酸的多肽，而不是全长的717个氨基酸的野生型聚合酶。为了支持基因组分析，在129X1/SVJ小鼠的睾丸提取液中没有检测到Poliota蛋白，而野生型Poliota通常是高表达的。Poliota的早期截断发生在该聚合酶的任何结构域合成之前，因此，我们推断129个衍生品系的小鼠应该被认为是Poliota活性的功能缺陷。然而，最近有报道称，在129个衍生菌株的Poli mRNA成熟过程中，外显子2有时被跳过，合成了一个675个氨基酸的无外显子2的Pol1蛋白，在体内外保持了催化活性。从结构的角度来看，我们发现这一想法是站不住脚的，因为外显子2编码的氨基酸包括对催化所需的金属离子的配位至关重要的残基，以及DNA聚合酶的结构完整性。为了确定外显子2缺失的Poliota异构体在体外是否具有催化活性，我们从杆状病毒感染的昆虫细胞中纯化了谷胱甘肽标记的全长外显子2(675个氨基酸)Poliota蛋白，并在体外将其与全长(717个氨基酸)GST标记的野生型小鼠Poliota进行了活性比较。在一定范围的镁或锰浓度以及不同的模板序列环境下进行反应条件。野生型小鼠POLIOTA表现出与人类POLIOTA生化特性相关的强健特征特性。然而，在相同的反应条件下，我们没有检测到任何与外显子2缺失的聚合酶相关的聚合酶活性，并得出结论，外显子2缺失的聚合酶蛋白在体外确实是催化不活跃的。

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.