Human DNA Polymerase N (POLN) Is a Low Fidelity Enzyme Capable of Error-free Bypass of 5S-Thymine Glycol

Abstract

Human DNA polymerase N (POLN or pol ) is the most recently discovered nuclear DNA polymerase in the human genome. It is an A-family DNA polymerase related to Escherichia coli pol I, human POLQ, and Drosophila Mus308. We report the first purification of the recombinant enzyme and examination of its biochemical properties, as a step toward understanding the functions of POLN. Unusual for an A-family DNA polymerase, POLN is a low fidelity enzyme incorporating T opposite template G with a frequency of 0.45 and G opposite template T with a frequency of 0.021. The frequency of misincorporation of T opposite template G is higher than any other known DNA polymerase. POLN has a processivity of DNA synthesis (1-100 nucleotides) similar to the exonuclease-deficient Klenow fragment of E. coli pol I, is inhibited by dideoxynucleotides, and resistant to aphidicolin. The strand displacement activity of POLN was higher than exonuclease-deficient Klenow fragment. Furthermore, POLN can perform translesion synthesis past thymine glycol, a common endogenous and radiationinduced product of reactive oxygen species damage to DNA. Thymine glycol blocks DNA synthesis by most DNA polymerases, but POLN was particularly adept at efficient and accurate translesion synthesis past a 5S-thymine glycol.The human genome contains 15 distinct known DNA polymerase genes, and these are classified into four families A, B, X, and Y based on their amino acid sequences (1). Human DNA polymerase N (POLN), 2 the most recently discovered nuclear DNA polymerase, is an A-family enzyme with unknown function. The gene on chromosome 4p16.2 encodes a protein of 900 amino acid residues with a molecular mass of 100 kDa (2). The prototypical A-family DNA polymerase, Escherichia coli pol I is a high fidelity DNA polymerase that contributes to the maturation of Okazaki fragments during DNA replication and in gap-filling during base excision repair (BER), nucleotide excision repair (NER), and repair of DNA interstrand cross-links.Human POLQ, another A-family DNA polymerase, is similar to the Drosophila nuclear DNA polymerase Mus308 (3) in that it encodes both a DNA/RNA helicase domain and an A-family DNA polymerase domain (4, 5). By contrast, POLN has only the DNA polymerase domain. The POLN gene is encoded only in vertebrate genomes, but not in invertebrates or any lower eukaryotes. Possibly POLN has a role related to organ systems that are especially developed in vertebrates, such as the adaptive immune system or the brain. Expression studies of POLN are limited, but expression of the gene has been detected by Northern blotting in testes, heart and skeletal muscle tissue (2) and by expression sequence tagging in prostate, muscle, brain, and other organs. In cells from a human neuroblastoma patient, a chromosome fusion (1, 4) disrupting the DNA polymerase domain coding sequence of POLN was observed at diagnosis and at relapse. A (4, 17) fusion was detected at relapse only (6). It is possible that POLN might serve as a tumor suppressor in some cell types and that loss...

Rapid induction of chromatin-associated DNA mismatch repair proteins after MNNG treatment

“…Additionally, cells undergoing continuous DTB while exposed to MNNG do not appear to incorporate BrdU, whereas cells undergoing continuous aphidicolin block while exposed to MNNG do appear to incorporate BrdU at a low level. Taken together these results indicate that DNA repair synthesis is occurring in aphidicolin blocked cells, perhaps by the BER pathway and nonreplicative polymerases uninhibited by aphidicolin [46,57,58].…”

A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol

“…Recent data have shown that RB69 gp43 is blocked by both cis stereoisomers of Tg, (5R, 6S) and (5S, 6R), whereas several DNA polymerases of the A family are blocked by (5R, 6S) Tg but are able to bypass the (5S, 6R) isomer (34). In the (5S, 6R) isomer, the configuration at the C5 position is such that the methyl group would protrude axially toward the adjacent 3Ј base.…”

Base Excision Repair and Lesion-Dependent Subpathways for Repair of Oxidative DNA Damage

“…Tg was originally identified in purified DNA after oxidation with ionizing radiation (29). The Tg lesion is a block to human replicative DNA polymerases, but the lesion is tolerated because several translesion DNA polymerases readily bypass the lesion, including DNA polymerase Z, k, n, b, and l (6,28,50,84), with a minimal increase in mutations. Additional oxidized thymine analogues include 5,6-dihydro-thymine (dHT) and 5-hydroxy-5,6-dihydrothymine (Th5) (Fig.…”