poliota, a remarkably error-prone human DNA polymerase

Abstract

The Saccharomyces cerevisiae RAD30 gene encodes DNA polymerase eta. Humans possess two Rad30 homologs. One (RAD30A/POLH) has previously been characterized and shown to be defective in humans with the Xeroderma pigmentosum variant phenotype. Here, we report experiments demonstrating that the second human homolog (RAD30B), also encodes a novel DNA polymerase that we designate poliota. poliota, is a distributive enzyme that is highly error-prone when replicating undamaged DNA. At template G or C, the average error frequency was approximately 1 x 10(-2). Our studies revealed, however, a striking asymmetry in misincorporation frequency at template A and T. For example, template A was replicated with the greatest accuracy, with misincorporation of G, A, or C occurring with a frequency of approximately 1 x 10(-4) to 2 x 10(-4). In dramatic contrast, most errors occurred at template T, where the misincorporation of G was, in fact, favored approximately 3:1 over the correct nucleotide, A, and misincorporation of T occurred at a frequency of approximately 6.7 x 10(-1). These findings demonstrate that poliota is one of the most error-prone eukaryotic polymerases reported to date and exhibits an unusual misincorporation spectrum in vitro.

Figure 1

(A) Purity of wild-type and mutant forms of GST-tagged polι. Purified wild-type and mutant GST-tagged polι fusion proteins (∼250 ng) was run on a 4%–20% polyacrylamide–SDS gel and visualized after staining with Coomassie brilliant blue R-250. (S) 10 kD molecular weight protein ladder (Life Technologies, MD); the migration of the 20, 40, 60, and 100 kD markers are indicated at left; (wt) wild-type GST–polι preparation used in the replication experiments; (m) D126A–E127A double alanine substituted mutant GST–polι protein. (B) DNA polymerase activity of the purified proteins. The position of the unextended radiolabeled primer (*P) as well as the products (P + 1, P + 2, etc.) are noted at the side of the figure. Reaction mixes were incubated at 37°C for 30 min and assays were performed in the absence of dNTPs (0) or in the presence of all four dNTPs (4). The lack of any discernable primer degradation in the absence of dNTPs suggests that polι does not possess any 3′–5′ exonucleolytic activity.

Figure 2

Ability of polι to incorporate nucleotides at various template sites. The extent of polι-dependent (mis)incorporation was measured at each template site in the absence (0) or presence of all four dNTPs (4) or presence of each individual dNTP (100 μm) (G, A, T, C). Reactions were for 30 min at 37°C. The sequence context of each template is given above each panel. In particular, note the very efficient misincorporation of G opposite T.

Figure 3

Kinetic analysis of polι-dependent (mis)incorporation at template T and template A. The ability of polι to incorporate the correct or incorrect nucleotide at both template sites was assayed at varying concentrations of dNTPs in a 5-min reaction, to allow a direct visual comparison of (mis)insertion fidelity. In most cases, the dNTP concentration varied from 0.1 to 100 μm. The exception was the incorporation of dTTP opposite template A, where the range was 1 nm to 1 μm. The sequence context of each template is given above each respective panel.

Figure 4

polι is a distributive enzyme. The ability of polι to extend the primer annealed to the wild-type template or the poly(A) template was assayed over a range of primer/template to enzyme concentrations. The primer/template was kept fixed at 20 nm and the enzyme varied from 40 nm to 0.4 nm as indicated. Reactions contained all four dNTPs (100 μm each) and were performed for 30 min at 37°C.