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. 2000 Oct 2;19(19):5259-66.
doi: 10.1093/emboj/19.19.5259.

Misinsertion and Bypass of Thymine-Thymine Dimers by Human DNA Polymerase Iota

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Misinsertion and Bypass of Thymine-Thymine Dimers by Human DNA Polymerase Iota

A Tissier et al. EMBO J. .
Free PMC article

Abstract

Human DNA polymerase iota (pol(iota)) is a recently discovered enzyme that exhibits extremely low fidelity on undamaged DNA templates. Here, we show that poliota is able to facilitate limited translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). More importantly, however, the bypass event is highly erroneous. Gel kinetic assays reveal that pol(iota) misinserts T or G opposite the 3' T of the CPD approximately 1.5 times more frequently than the correct base, A. While pol(iota) is unable to extend the T.T mispair significantly, the G.T mispair is extended and the lesion completely bypassed, with the same efficiency as that of the correctly paired A. T base pair. By comparison, pol(iota) readily misinserts two bases opposite a 6-4 thymine-thymine pyrimidine-pyrimidone photoproduct (6-4PP), but complete lesion bypass is only a fraction of that observed with the CPD. Our data indicate, therefore, that poliota possesses the ability to insert nucleotides opposite UV photoproducts as well as to perform unassisted translesion replication that is likely to be highly mutagenic.

Figures

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Fig. 1. polι-dependent replication of undamaged, CPD-containing and 6-4PP-containing DNA templates. Reactions were performed as described in Materials and methods for the times noted below each track. The template sequence is indicated at the top of the figure, and replication products (P+1, P+2, etc.) are indicated at the right of (A). (A) Replication promoted by wild-type GST–polι. (B) Replication in the presence of a mutant (D126A/E127A) GST–polι devoid of polymerase activity (Tissier et al., 2000). In these experiments, reactions were performed for 30 min in the presence of an undamaged template (UN), a CPD template (CPD) or a 6-4PP-containing template (6-4).
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Fig. 2. Incorporation of nucleotides opposite a CPD or 6-4PP. The sequence of each template is indicated above each panel. Each primer/template was incubated for 30 min with polι in the absence of dNTPs (0), all four dNTPs (4), or each dNTP individually (G, A, T, C). The size of replication products (P+1, P+2, etc.) is indicated at the right and left of the figure.
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Fig. 3. Steady-state kinetic analysis of polι-dependent nucleotide incorporation opposite the 3′ T of a CPD or 6-4PP. (A) CPD. (B) 6-4PP. The concentration of each nucleotide (in µM) added to the reaction is indicated below each track. Experiments illustrating incorporation opposite the CPD were performed for 10 min, while those showing polι-dependent incorporation opposite the 6-4PP were for 2 min.
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Fig. 4. polι-dependent extension of primers opposite the 3′ T of the CPD or the 6-4PP. The 3′ sequence of each primer is shown above each group of experiments. (A) CPD. (B) 6-4PP. 0, 4, G, A, T and C indicate reactions in the absence of nucleotides, all four nucleotides, or G, A, T or C, respectively.
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Fig. 5. polι-dependent extension of primers with various 3′ dinucleotides opposite the CPD or 6-4PP. Each reaction was performed for 15 min in the absence of nucleotides (0), or all four nucleotides (4). The 3′ dinucleotide sequence of each primer is given below each panel. The left panel shows extension from the CPD-containing template, while on the right are extensions from the 6-4PP-containing template. The track in the middle, labeled UN, demonstrates replication from an ‘AA’ primer and an undamaged template. Note that any extension from these primers represents complete bypass of the respective lesion.
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Fig. 6. Possible mechanisms of translesion replication in wild-type and XP-V cells. In both cases, a CPD is encountered by the cell’s main replicase, polδ (shown in yellow). In a normal cell, translesion replication is accurately performed by polη (shown in light blue). This is clearly the main replicative pathway utilized by the cell to bypass a CPD and we have indicated this fact by larger sized arrows and labeling. On occasions, however, polη misinserts a base opposite the 3′ T of the CPD (Johnson et al., 2000; Masutani et al., 2000). These mispairs cannot be extended by polη, but are probably extended by polζ (shown in light green). Alternatively, polζ may perform unassisted translesion replication, which is error prone. This model incorporates a central role in error-prone lesion bypass attributed to polζ and explains why overproduction of antisense to hRev3 (the catalytic cores of polζ) results in a dramatic reduction of UV light-induced mutagenesis in human cells (Gibbs et al., 1998). In XP-V cells devoid of polη, lesion bypass is highly error prone and probably occurs as a consequence of three discrete pathways: unassisted polι-dependent bypass (polι is shown in pink); polι-misincorporation followed by polζ extension of the mispair; or unassisted polζ-dependent bypass. If the two polι-dependent pathways predominate over the unassisted polζ-bypass pathway, it would explain why the spectra of mutations are very different in wild-type and XP-V cell lines (Wang et al., 1993; McGregor et al., 1999). In all cases, polη, polι or polζ only replicates a few nucleotides surrounding the lesion before dissociating from the primer/template and is rapidly replaced by polδ, which completes chromosome duplication.

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