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，包括对细菌、古生菌和真核生物的研究。 在过去的一年里，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715个氨基酸的蛋白质。 DNA测序技术的进步使得人们认识到，存在上游的、符合读框的起始密码子，其将编码740个氨基酸的DNA聚合酶i（pol i ota）蛋白。 额外的25个氨基酸区域富含酸性残基（11/25），并且在从鱼到人的真核生物中合理地保守。 因此，策展参考序列（RefSeq）数据库将pol-iota鉴定为740个氨基酸的蛋白质。 然而，740个氨基酸的pol iota蛋白的存在从未被实验证明。 使用针对pol iota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更长的740个氨基酸（iota-长）同种型。 然而，通过免疫沉淀富集后检测到痕量的iota-长同种型。有人可能会争辩说，较长的同种型可能具有独特的生物学功能，如果它表现出显着的差异，其酶性质从较短的，充分表征的715个氨基酸的多聚体。 因此，我们纯化并表征了重组全长（740个氨基酸）长多聚体，并在体外将其与全长（715个氨基酸）短多聚体进行了比较。 最佳催化活性的金属离子要求在长聚物和短聚物之间略有不同，但在最佳条件下，两种同种型在体外表现出不可区分的酶性质。 我们还报道了与短聚体一样，长聚体同种型在体内可以被单泛素化和多泛素化，以及在体内形成损伤诱导的病灶。 我们的结论是，人类细胞中的主要同种型是较短的715个氨基酸的蛋白质，并且如果或当表达时，较长的740个氨基酸的同种型具有与显著更丰富的较短同种型相同的性质。 在2003年，我们报道了129衍生的小鼠品系在Poli基因的密码子27处携带天然存在的无义突变，其将产生仅26个氨基酸的poliota肽，而不是全长717个氨基酸的野生型聚合酶。 为了支持基因组分析，在来自129 X1/SvJ小鼠的睾丸提取物中未检测到pol i ota蛋白，其中野生型pol i ota通常高度表达。 在聚合酶的任何结构域被合成之前，聚合酶中的早期截短发生，因此，我们推断129衍生的小鼠品系应被认为在聚合酶活性方面有功能缺陷。 然而，最近有报道称，在129衍生菌株中Poli mRNA的成熟过程中，外显子-2有时被跳过，并且合成了一种675个氨基酸的外显子-2较少的pol蛋白，其在体外和体内保留了催化活性。 从结构的角度来看，我们发现这种想法是站不住脚的，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以及DNA聚合酶的结构完整性至关重要的残基。 为了确定无外显子2的poliota同种型是否具有体外催化活性，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无外显子2（675个氨基酸）poliota蛋白，并在体外将同种型的活性与全长（717个氨基酸）GST标记的野生型小鼠poliota进行比较。 反应条件在一定范围的镁或锰浓度以及不同的模板序列背景下进行。 野生型小鼠多聚体表现出先前与人多聚体生物化学性质相关的稳健特征性质。 然而，在相同的反应条件下，我们没有检测到与外显子-2-少的聚合酶相关的任何聚合酶活性，并得出结论，外显子-2-少的聚合酶蛋白在体外确实是无催化活性的。

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.