Fidelity and Processivity of DNA Synthesis by DNA Polymerase κ, the Product of the Human DINB1 Gene

Abstract

Mammalian DNA polymerase (pol ), a member of the UmuC/DinB nucleotidyl transferase superfamily, has been implicated in spontaneous mutagenesis. Here we show that human pol copies undamaged DNA with average single-base substitution and deletion error rates of 7 ؋ 10 ؊3 and 2 ؋ 10 ؊3 , respectively. These error rates are high when compared to those of most other DNA polymerases. pol also has unusual error specificity, producing a high proportion of T⅐CMP mispairs and deleting and adding non-reiterated nucleotides at extraordinary rates. Unlike other members of the UmuC/ DinB family, pol can processively synthesize chains of 25 or more nucleotides. This moderate processivity may reflect a contribution of C-terminal residues, which include two zinc clusters. The very low fidelity and moderate processivity of pol is novel in comparison to any previously studied DNA polymerase, and is consistent with a role in spontaneous mutagenesis.The recently discovered UmuC/DinB nucleotidyl transferase superfamily of DNA polymerases (1-3) includes a subfamily whose members share extensive amino acid sequence homology with the Escherichia coli dinB gene product. The dinB gene is required for untargeted mutagenesis of phage (4), and overexpression of dinB in E. coli increases the spontaneous mutation rate in plasmids, especially for single-base deletions in a run of guanine residues (5). The dinB gene encodes DNA polymerase (pol) IV, a distributive enzyme that lacks detectable 3Ј35Ј exonuclease activity (6). pol IV has limited ability to bypass UV radiation-induced photoproducts, but misinserts nucleotides at undamaged template sites at rates that are higher than those observed for DNA pol III, the major replicative enzyme in E. coli (7). Moreover, when incubated in the presence of a template-primer with a terminal mismatch with a potential for misalignment, pol IV generates DNA products that are one nucleotide shorter than expected (6). This is consistent with the Ϫ1 frameshift mutations seen when dinB is overexpressed (5).Orthologs of E. coli dinB have been identified in eukaryotes (2, 8). The human DINB1 gene is localized at chromosome 5q13 and encodes an 870-amino acid DNA polymerase (9 -11), which we refer to here as DNA polymerase (pol ).1 The product of the hDINB1 gene has also been designated DNA polymerase (10), a designation used earlier (12) for the human homolog of the Drosophila melanogaster mus308 gene. pol has several properties in common with E. coli pol IV. When purified from insect cells expressing the full-length polymerase fused to glutathione S-transferase (GST) (11) or purified as a catalytically active fragment of amino acids 1-560 (9), pol lacks detectable 3Ј35Ј exonuclease activity. The purified full-length GST fusion protein has optimal activity at 37°C over the pH range 6.5-7.5, it is insensitive to inhibition by aphidicolin or dideoxynucleotides, and Mg 2ϩ is preferred over Mn 2ϩ as the essential divalent cation (11). Neither the full-length GST-enzyme purified from yeast cells (10) nor truncated pol (9)...

DNA polymerases in adaptive immunity

“…As Polκ does not seem to play a role in the physiological situation 63,64 , it suggests that this polymerase acts as a back-up when the normal pathway is compromised. Accordingly, the reduction in A/T mutagenesis corresponds well with the lower mutagenicity of Polκ 61,62 . Altogether these data support the proposition that Polη is the sole contributor of A/T mutations during physiological SHM in mice, with other TLS polymerases playing a backup role 65 .…”

“…Half of these mutations consist in A to C and T to G transversion mutations, a pattern that evokes the signature of another TLS enzyme, Polκ 61,62 (Fig.4). As Polκ does not seem to play a role in the physiological situation 63,64 , it suggests that this polymerase acts as a back-up when the normal pathway is compromised.…”

8-(Hydroxymethyl)-3,N4-etheno-C, a Potential Carcinogenic Glycidaldehyde Product, Miscodes In Vitro Using Mammalian DNA Polymerases

“…Human pol κ synthesizes undamaged DNA with very low fidelity (21,22). A recent lung tumor study implicates the overexpression of pol κ in tumorigenesis (23), which may account for the accumulation of mutations as the enzyme has very low fidelity.…”

“…Polymerase η (pol η) is coded by the XP-V gene and can bypass cis-syn thymine dimers in an error-free 9/3/01 manner. Polymerase κ (pol κ), the hDINB1 gene product, is a newly identified enzyme with low fidelity and has both error-free and error-prone activities in DNA lesion bypass (20)(21)(22). More recently, O-Wang et al (23) reported the overexpression of pol κ in patients with non-small cell lung cancer.…”

Proficient and Accurate Bypass of Persistent DNA Lesions by DinB DNA Polymerases

“…DinB from E. coli synthesizes undamaged and certain damaged templates with error frequencies between 10 -3 and 10 -5 , 11,28 while the mammalian ortholog synthesizes undamaged DNA with a slightly higher error frequency of 10 -2 -10 -3 . 29 Both enzymes also have the ability to produce -1 frameshift mutations during DNA synthesis at an appreciable frequency. 8,28,29 Despite these error frequencies, certain data suggest that DinB/pol k function may in fact be antimutagenic in vivo.…”