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. 2007 Mar 1;402(2):321-9.
doi: 10.1042/BJ20060898.

Accessory Proteins Assist Exonuclease-Deficient Bacteriophage T4 DNA Polymerase in Replicating Past an Abasic Site

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Accessory Proteins Assist Exonuclease-Deficient Bacteriophage T4 DNA Polymerase in Replicating Past an Abasic Site

Giuseppina Blanca et al. Biochem J. .
Free PMC article

Abstract

Replicative DNA polymerases, such as T4 polymerase, possess both elongation and 3'-5' exonuclease proofreading catalytic activities. They arrest at the base preceding DNA damage on the coding DNA strand and specialized DNA polymerases have evolved to replicate across the lesion by a process known as TLS (translesion DNA synthesis). TLS is considered to take place in two steps that often require different enzymes, insertion of a nucleotide opposite the damaged template base followed by extension from the inserted nucleotide. We and others have observed that inactivation of the 3'-5' exonuclease function of T4 polymerase enables TLS across a single site-specific abasic [AP (apurinic/apyrimidinic)] lesion. In the present study we report a role for auxiliary replicative factors in this reaction. When replication is performed with a large excess of DNA template over DNA polymerase in the absence of auxiliary factors, the exo- polymerase (T4 DNA polymerase deficient in the 3'-5' exonuclease activity) inserts one nucleotide opposite the AP site but does not extend past the lesion. Addition of the clamp processivity factor and the clamp loader complex restores primer extension across an AP lesion on a circular AP-containing DNA substrate by the exo- polymerase, but has no effect on the wild-type enzyme. Hence T4 DNA polymerase exhibits a variety of responses to DNA damage. It can behave as a replicative polymerase or (in the absence of proofreading activity) as a specialized DNA polymerase and carry out TLS. As a specialized polymerase it can function either as an inserter or (with the help of accessory proteins) as an extender. The capacity to separate these distinct functions in a single DNA polymerase provides insight into the biochemical requirements for translesion DNA synthesis.

Figures

Figure 1
Figure 1. DNA substrates used in the present study
X represents the position of the artificial abasic site (tetrahydrofuran residue). (A) linear 60/17-mer substrate; (B) linear 60/17-mer substrate containing the abasic site; (C) circular 100/17-mer substrate; (D) circular 100/17-mer substrate containing an abasic site for running start experiments; (E) circular 100/18-mer substrate containing an abasic site for standing start experiments.
Figure 2
Figure 2. Replication of linear DNA substrates by gp43 wt and gp43D219A exo DNA polymerases
(A) PhosphorImage of the reaction products for undamaged substrate, A, or substrate that contains an AP lesion. Reactions were performed as described in the Materials and methods section with 30 nM substrates A or B for 30 min at 37 °C at the indicated concentrations of polymerases. In the case of lanes 5–8 and 9–12, concentrations of the enzymes were 0.01 nM, 0.1 nM, 1 nM and 10 nM respectively. The position of the 17-mer (primer) and of the 60-mer (full-length product) are indicated on the left-hand side of the gel, while the location of the abasic site is indicated on the right-hand side. (B) Quantification of the data shown in (A) for the damaged substrate.
Figure 3
Figure 3. Replication of circular DNA substrates by gp43 wt and gp43D219A exo DNA polymerases in a running start reaction
(A) PhosphorImage of the reaction products. Reactions were performed as described in the Materials and methods section with 15 nM of running start substrates C (undamaged) or D (with an AP lesion) for 30 min at 37 °C at the indicated concentrations of polymerases. In the case of lanes 6–10 and 11–15, concentrations of the enzymes were 0.001 nM, 0.01 nM, 0.1 nM, 1 nM and 10 nM respectively. The position of the 17-mer (primer) and of the 100-mer (full-length product) are indicated on the left-hand side of the gel, while the location of the abasic site is indicated on the right-hand side. (B) Quantification of the data shown in (A) for the damaged substrate.
Figure 4
Figure 4. Insertion of a nucleotide in front of the lesion of an AP-containing circular DNA substrate in a running start reaction
(A) PhosphorImage of the reaction products. Running start reactions were performed as described in the Materials and methods section with 15 nM substrate D at the indicated times and concentrations of polymerases. The position of the 17-mer (primer) is indicated on the left-hand side of the gel while the location of the abasic site is indicated on the right-hand side. (B) Quantification of the data shown in (A).
Figure 5
Figure 5. Replication of the AP-containing circular DNA substrate by gp43wt and gp43D219A exo DNA polymerases, in combination with other replicative proteins, in a running start reaction
(A) PhosphorImage of the reaction products. Reactions were performed as described in the Materials and methods section with 15 nM running start substrate D, 0.05 nM of either gp43 wt or gp43D219A exo polymerases, 100 nM of gp45, 200 nM of gp44/62 and 150 nM of gp32. The incubation time was 30 min at 37 °C. The position of the 17-mer primer is indicated on the left-hand side of the gel while the location of the abasic site and of the full-length 100-mer product are indicated on the right-hand side. Lane 14 is the same as lane 13, except that ATP has been omitted from the reaction mixture. (B) Quantification of the extension from the AP site by the D219A exo polymerase, either alone or in combination with the replicative proteins. Data are the average of three independent experiments±S.E.M.
Figure 6
Figure 6. Replication of the AP-containing circular DNA substrate by gp43wt and gp43D219A exo DNA polymerases, in combination with other replicative proteins, in both running and standing start reactions
(A) Reactions were performed as described in the Materials and methods section with 15 nM running start or standing start substrate D, 0.05 nM of either gp43 wt or gp43D219A exo polymerases, 100 nM of gp45, 200 nM of gp44/62 and 150 nM of gp32. The incubation time was 30 min at 37 °C. The position of the 17-mer primer, the AP site and the 100-mer full-length product for the running start reaction are indicated on the left-hand side of the gel, while the location of the 18-mer primer and of the abasic site for the standing start reaction are indicated on the right-hand side. (B) Quantification of the extension from the AP site by the D219A exo polymerase, either alone or in combination with the replicative proteins. Data are the average of four independent experiments±S.E.M.
Figure 7
Figure 7. Model for DNA replication by T4 polymerase based on structural data for the homologous DNA polymerase RB69 [–32]
In this scheme T4 DNA polymerase (Pol), which has a polymerase catalytic site (p) and an exonuclease catalytic site (e), binds to an undamaged P/T DNA with base n at the coding position on the template strand (P/Tn) to form a binary complex (Bn). Pol and Bn are in an open conformation indicated as a sphere and P/Tn is situated in the exonuclease site of the binary complex (see text). Binding of complementary substrate dNTP (N) to Bn induces a closed ternary complex (Tn) depicted as an ellipse. During this conformational change the P/T DNA rotates 40 ° and its extremity moves 40 Å (1 Å=0.1 nm) so that N, n and the 3′-OH terminus of the primer strand are at p and the enzyme catalyses polymerization (ke). Addition of N to the primer strand is followed by pyrophosphate (PPi) release and translocation. Subsequent nucleotide addition that leads to elongation probably occurs via a closed polymerase complex, designated [], whose structure has not yet been determined. The polymerase complex attains thermodynamic equilibrium between each nucleotide addition step [36] and consequently can either dissociate or bind the following dNTP and enter the next elongation cycle.

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