POLN, a Nuclear PolA Family DNA Polymerase Homologous to the DNA Cross-link Sensitivity Protein Mus308

Federica Marini,Nayun Kim,Anthony Schuffert,Richard D Wood

doi:10.1074/jbc.m305646200

Federica Marini, Nayun Kim + Show 2 more

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https://doi.org/10.1074/jbc.m305646200

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Abstract

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.

Highlights

A growing number of mammalian DNA polymerases have been identified within recent years, and initial characterization suggests that they have specialized roles in DNA replication or repair
We report here the identification, cloning, and characterization of POLN and its gene product, a new mammalian DNA polymerase related to Mus308
Isolation of a Novel A-family DNA Polymerase Gene from Mammalian Genomes—Searching for mammalian homologs of E. coli DNA pol I, we found a mouse expressed sequence tag clone (AA475002) and a human sequence (AF044578) related to pol I but unlinked to POLQ

Summary

Introduction

A growing number of mammalian DNA polymerases have been identified within recent years, and initial characterization suggests that they have specialized roles in DNA replication or repair. An apparent human ortholog of Mus308, designated POLQ or DNA pol ␪, is encoded in the human genome (NCBI accession number NM_006596), and a cDNA representing the COOH-terminal part of the gene has been isolated [3]. We recently isolated a mammalian DNA helicase gene designated HEL308, which is homologous to the NH2-terminal portion of Drosophila Mus308 [4]. In E. coli, ICLs can be repaired by the coordinated action of nucleotide excision repair and homologous recombination [6, 7]. In this pathway, the prototype A family enzyme pol I plays a crucial role

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