Marini Federica, Kim Nayun, Schuffert Anthony, Wood Richard D
University of Pittsburgh Cancer Institute, Hillman Cancer Center, Research Pavilion, Pittsburgh, Pennsylvania 15213, USA.
J Biol Chem. 2003 Aug 22;278(34):32014-9. doi: 10.1074/jbc.M305646200. Epub 2003 Jun 6.
The Drosophila Mus308 gene is unusual in encoding both a family A DNA polymerase domain and a DNA/RNA helicase domain. A mus308 mutation was shown to result in increased sensitivity to DNA cross-linking agents, leading to the hypothesis that Mus308 functions in the repair of DNA interstrand cross-links. Recently a mammalian ortholog of Mus308, POLQ, has been identified. We report here the identification, cloning, and characterization of POLN and its gene product, a new mammalian DNA polymerase also related to Mus308. The human cDNA encodes a protein of 900 amino acid residues. The region starting from residue 419 shares 33% identity (48% similarity) with the equivalent region of Escherichia coli DNA polymerase I. POLN is expressed in human cell lines with numerous alternatively spliced transcripts, and a full-length human coding region that comprises 24 exons within 160 kilobases of genomic DNA. Expression analysis by northern blotting and in situ hybridization showed highest expression of full-length POLN in human and mouse testis. POLN localized to the nucleus when expressed as a enhanced green fluorescent protein (GFP)-tagged protein in human fibroblasts. GFP-tagged recombinant POLN had DNA polymerase activity on activated calf thymus DNA and on a singly primed template.
果蝇的Mus308基因不同寻常，它编码一个A家族DNA聚合酶结构域和一个DNA/RNA解旋酶结构域。研究表明，mus308突变会导致对DNA交联剂的敏感性增加，从而提出Mus308在DNA链间交联修复中发挥作用的假说。最近，已鉴定出Mus308的哺乳动物直系同源物POLQ。我们在此报告POLN及其基因产物的鉴定、克隆和特征，POLN是一种也与Mus308相关的新型哺乳动物DNA聚合酶。人类cDNA编码一个由900个氨基酸残基组成的蛋白质。从第419位残基开始的区域与大肠杆菌DNA聚合酶I的等效区域具有33%的同一性（48%的相似性）。POLN在人类细胞系中表达，有许多可变剪接转录本，其全长人类编码区在160千碱基的基因组DNA内包含24个外显子。通过Northern印迹和原位杂交进行的表达分析表明，全长POLN在人类和小鼠睾丸中表达最高。当在人类成纤维细胞中作为增强型绿色荧光蛋白（GFP）标记的蛋白表达时，POLN定位于细胞核。GFP标记的重组POLN对活化的小牛胸腺DNA和单引物模板具有DNA聚合酶活性。
