We report the expression, purification and characterization of recombinant human Pol δ wild-type and exonuclease-mutant enzymes. Several laboratories have already reported recombinant human Pol δ prepared by various systems. Podust et al
) and Xie et al
) independently developed baculoviral vectors for human Pol δ expression. These systems provided high yields of the active enzyme that may be subject to post-translational modifications specific for eukaryotes. However, since transfer of recombinant DNAs to baculovirus is a time-consuming step, the baculoviral expression systems may not be convenient for preparation of numerous mutant enzymes. Recently, Masuda et al
) reported bacterial expression and purification of untagged human Pol δ. Their system also provided a high yield of active enzyme, but its purification included two column chromatography steps with linear gradient elution and one size-exclusion column step. The expression and purification system for human Pol δ described here is less laborious than the others and suitable for rapid screening of mutant constructs.
The exonuclease activity of Pol δ has been well studied in yeast systems. Jin et al
) reported that S. cerevisiae
Pol δ has a strong exonuclease activity which can completely degrade a primer annealed to a template within 10 min in the absence of dNTPs. In contrast, the exonuclease activity of S. pombe
reported by Chen et al
) appears to be much milder than that of S. cerevisiae
. The human Pol δ described here did not extensively degrade primers annealed to templates in the absence of dNTPs (, lanes with the wild-type enzyme). This seems to be consistent with the relationship between humans and two yeasts in which humans are biologically closer to S. pombe
than S. cerevisiae
. To quantitatively detect the exonuclease activity of human Pol δ, we employed a single-stranded oligonucleotide as a substrate for exonuclease assay (C). This assay demonstrated that the wild-type human enzyme indeed has 3′ → 5′ exonucleae activity. Furthermore, the mutant enzymes in which the conserved exonuclease domain was altered decreased their exonuclease activities by more than 95% compared to the wild type. These results indicate that the ssDNA exonuclease activity is derived from the conserved exonuclease domain of human Pol δ.
In spite of the apparently weak exonuclease activity on dsDNA, the human Pol δ showed significant proofreading activities on 3′-mispaired primers (B). In the same assay, three exonuclease-mutant enzymes showed constantly lower proofreading activities than the wild-type enzyme. Nevertheless, comparison of the mispair-extension and proofreading activities by the wild-type and mutant enzymes on a series of 3′-mispaired primer/template combinations suggest that not only mispaired nucleotides but also their surrounding sequences and/or distances from the primer's 5′ terminus may affect Pol δ's behavior when encountering a 3′-mispair. Using a more simple enzyme, Bacillus stearothermophilus
DNA polymerase I large fragment, Johnson and Beese (36
) analyzed all 12 possible mispairs, and accordingly categorized into four groups of mispairs (as shown as primer nucleotide:template nucleotide): (i) G:T, G:G, A:C, T:C to disrupt template strand and pre-insertion site; (ii) T:T, C:T to disrupt primer strand assembly of catalytic site; (iii) A:G, T:G to disrupt template and primer strands; (iv) A:A, G:A, C:C to be frayed at insertion site. However, our results do not indicate similarity among data with 57 nt G:G, 59 nt T:C and 63 nt G:G while data with 70 nt A:A and 71 nt C:C appear similar (B). Johnson and Beese also proposed the effect of long-range distortions caused by a mismatch up to 6 nt away from the primer end (36
). Thus further analyses with a large collection of mispaired primer/template designed by a more systematic manner will be required to decipher universal rules governing mispair extension and proofreading by DNA polymerase.
Identification of the deletion products from the 57 nt G:G primer extension directly uncover some flexibility of human Pol δ in the pocket wherein the template/primer DNA resides. It should be noticed, however, that the experimental condition we employed may have allowed formation of the loop-out before the enzyme was assembled to DNA for polymerization. Therefore it is not clear whether the 3′-mispair can be converted inside the polymerase–DNA complex to the template loop-out.
For analyses of the proofreading activity and also for the lesion bypass analyses, we employed pyrosequencing to determine the nucleotide inserted into the fully extended product at the position of interest. This method enabled us to obtain quantitative data in a fast and cost-effective manner compared to the conventional method which requires subcloning of the products and subsequent sequencing of at least 20 clones. The pyrosequencing technique also has its own disadvantage, such as ambiguity derived from deletion, insertion and homonucleotide regions, relatively high background up to 10%. As described for the 57 nt G:G primer extension, analysis of more than two types of products may be further complicated. Nevertheless, the result obtained from pyrosequencing was in good agreement with that from conventional sequencing (B, ).
Translesion activity of Pol δ was tested with templates carrying either an 8-OG or AP site analog. DNA synthesis by Pol δ efficiently passed through the 8-OG, whereas the AP site analog was a relatively tight blocker. Haracska et al
) demonstrated that AP site cannot be bypassed by S. cerevisiae
Pol δ alone. In contrast, Mozzherin et al
) reported that PCNA increases AP site bypass by calf thymus Pol δ. In our system including PCNA and RFC, human Pol δ proceeded DNA synthesis through an AP site-containing template by 20–30% efficiency. This result suggests that while a majority of AP sites encountered by the replication machinery may be bypassed with help by TLS-type DNA polymerase(s), some part of AP site bypass can be carried out by Pol δ. Recently Liao et al
) characterized replication of AP site-containing DNA by Xenopus
egg extract under the condition in which the AP site repair was suppressed. In this study, resultant replication products were classified into three groups by nucleotides inserted opposite the AP site: (i) a nucleotide instructed by the complementary strand (error-free products); (ii) dAMP regardless of the complementary strand sequence (error-prone, A-rule); (iii) dCMP regardless of the complementary strand sequence (error-prone, C-rule). The result in the present study implies that some, if not all, of the error-prone products carrying A opposite the AP site could result from bypass by Pol δ.
It is a striking observation that one of the exonuclease-mutant Pol δ, exo-24, had a lower lesion-bypass activity than the wild-type. This unique character of exo-24 may be related to the severe inability to extend mispaired primers (). The higher proofreading efficiency in the products fully extended by the exo-24 enzyme compared to the other mutants (B) can result from its stringent extension activity. One possible explanation for the exo-24's salient property is that this mutated exonuclease domain may bind to the primer end more tightly than the wild-type and other mutants, leading to inefficient accessibility of the primer end by the polymerase domain. Hogg et al
) reported an RB69 polymerase mutant which affects active site switching, although this mutant preferentially hold the primer DNA in the polymerase active site. Our model on exo-24 could be tested by similar structural analysis.
Nucleotide preference of Pol δ to insert opposite 8-OG is the important factor in this lesion's mutagenicity. Shibutani et al
) demonstrated that calf thymus Pol δ with PCNA inserted C and A with a ratio of 1:5 opposite 8-OG. In addition, Haracska et al
) reported that S. cerevisiae
Pol δ predominantly inserted A opposite 8-OG. On the other hand, Maga et al
) reported that, when combined with PCNA ± RPA, human Pol δ inserted C and A by 3:1 ratio, suggesting that this lesion may be less mutagenic. Our data also indicated that human Pol δ slightly preferred C insertion to A insertion at this lesion. One possible reason for the apparent discrepancy in these observations is that replication auxiliary proteins may affect nucleotide selection as suggested by Maga et al
). Their data revealed that the addition of PCNA reverted Pol δ's nucleotide preference from mutagenic A to correct C. All the experimental systems discussed here except ours employed oligonucleotides with free ends as a primer/template and therefore PCNA may not be stably loaded on them. In contrast, our assay system employed end-blocked oligonucleotides, and PCNA and RFC were essential for primer extension by Pol δ in this system (B). Another possible reason for different results on 8-OG bypass is that it may due to different lengths of extension beyond the lesion. Most previous studies examined the nucleotide inserted opposite the lesion after limited lengths of extension (0–5 nt), whereas the reaction products we analyzed here resulted from extension of at least 30 nt after the lesion. While studies of steady-state kinetics on insertion and extension (7
) can dissect the lesion bypass reaction by DNA polymerase at a single nucleotide level, the distorted pair of the inserted nucleotide and the lesion may affect the extension at relatively distant positions as suggested with a study of mispaired primer extensions (36
). Our results seem to represent products preferred for long extension and simulate the natural lesion bypass by Pol δ itself. Previously we observed that A:8-OG mispair was repaired in the replication-dependent manner in MYH-deficient mouse cells at a significant efficiency although the wild-type cells repaired better (40
). Taking account of Pol δ's efficient bypassing of this lesion (), the replication-associated repair of the A:8-OG mispair in the absence of MYH may be explained by the suboptimal C insertion in translesion synthesis by Pol δ in vivo
, although partial contribution of TLS polymerases is not ruled out.