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1.
FIGURE 2.

FIGURE 2. From: Coordination between Polymerase ? and FEN1 Can Modulate CAG Repeat Expansion.

DNA synthesis of pol β in the context of CAG repeats and random DNA sequence. pol β primer extension DNA synthesis on substrates with CAG repeats (right panel) or random DNA sequence (left panel) was measured in the presence of 1 μm OGG1 and 50 nm APE1 as described under “Experimental Procedures.” Lanes 1 represents reaction mixture in the absence of pol β. Lanes 2 represents reaction mixture containing 2 nm pol β. Nucleotides inserted by pol β are indicated by arrows. Lane 3 represents synthetic DNA markers that mimic pol β nucleotide insertions of dAMP. 5′-32P-Labeled DNA substrate (25 nm) was used in each reaction mixture. The DNA substrate is illustrated schematically above the gel.

Yuan Liu, et al. J Biol Chem. 2009 October 9;284(41):28352-28366.
2.
FIGURE 5.

FIGURE 5. From: Coordination between Polymerase ? and FEN1 Can Modulate CAG Repeat Expansion.

Coordinated activities of pol β, FEN1, and DNA ligase prevent CAG repeat expansion. The role of coordination among BER enzymes in CAG repeat expansion was examined with a substrate containing a THF group embedded in (CAG)20 repeats. The DNA substrate is illustrated schematically above the gel. BER reactions were reconstituted with pol β (lanes 4 and 5), 5 nm FEN1, and 5 nm LIG I in the presence of dGTP and dCTP under the conditions described under “Experimental Procedures.” BER enzymes indicates a mixture of 5 nm LIG I and 5 nm FEN1. Lane 3 represents a reaction mixture in the presence of APE1 alone. Lane 2 represents substrate alone. Lane 1 (M) represents markers of 100, 115, and 130 nt. Unexpanded product and nucleotides inserted by pol β are indicated. The unexpanded product was verified by sizing analysis as described under “Experimental Procedures.” 5′-32P-Labeled DNA substrate (10 nm) was used.

Yuan Liu, et al. J Biol Chem. 2009 October 9;284(41):28352-28366.
3.
FIGURE 7.

FIGURE 7. From: Coordination between Polymerase ? and FEN1 Can Modulate CAG Repeat Expansion.

HMGB1 stimulates CAG repeat expansion mediated by APE1 and FEN1. A, the effects of HMGB1 on the CAG repeat expansion mediated by APE1 and FEN1 were examined in the presence of limiting APE1 (0.1 nm) along with 2 nm pol β, 100 units of T4 DNA ligase, and 10 nm HMGB1. B, the effect of HMGB1 on FEN1-mediated CAG repeat expansion was examined in the presence of limiting FEN1 (0.5 nm), along with 2 nm pol β, 100 units of T4 DNA ligase, and 10 nm HMGB1. The reaction mixtures were reconstituted in the absence and presence of 10 nm HMGB1 as described under “Experimental Procedures.” 10 nm 5′-32P-labeled substrate containing 20 CAG repeats was used. The guanine of the first CAG of the substrate was substituted by a THF residue, mimicking a modified, i.e. reduced or oxidized, abasic site as indicated by an arrow. C, stimulation of HMGB1 on the APE1 5′-incision activity on (CAG)20-THF was determined in the presence of 0.1 nm APE1 and 10 nm HMGB1 as described in “Experimental Procedures.” D, the effect of HMGB1 on FEN1 cleavage of 5′-THF-(CAG)20 hairpin was examined in the presence of 1 nm FEN1 and 10 nm HMGB1. The reaction mixtures were constituted as described under “Experimental Procedures.” Lane 1 represents incubation in the absence of HMGB1, whereas lane 2 corresponds to incubation in the presence of HMGB1. The substrates were 32P-radiolabeled at the 5′-end of the damage-containing strand (asterisk) and are illustrated schematically above the gels.

Yuan Liu, et al. J Biol Chem. 2009 October 9;284(41):28352-28366.
4.
FIGURE 6.

FIGURE 6. From: Coordination between Polymerase ? and FEN1 Can Modulate CAG Repeat Expansion.

HMGB1 facilitates CAG repeat expansion. Panels A and C include schematic representation of substrates A, cell extracts with and without HMGB1 were prepared from HMGB1 null (−/−) and wild type (+/+) mouse embryonic fibroblasts. Substrates (25 nm) were incubated with 60 μg of cell extract at 37 °C for 30 min as described in the legend for Fig. 1. Lanes 1 and 2 correspond to reaction mixtures containing cell extract from HMGB1 wild type (+/+) and null (−/−) cell lines, respectively. The expansion products and 100 nt of unexpanded product are indicated. A lane with DNA size markers is shown (M). B, quantification of the amount of expansion products. The blue bars represent expansion products resulting from the CAG repeat substrate. The red bar represents a background signal from reaction mixtures with random sequence substrate. C, the effect of HMGB1 on CAG repeat expansion was examined by adding purified HMGB1 to the HMGB1 null cell extract, and incubation was under the same conditions described in A. Size markers are shown.

Yuan Liu, et al. J Biol Chem. 2009 October 9;284(41):28352-28366.
5.
FIGURE 4.

FIGURE 4. From: Coordination between Polymerase ? and FEN1 Can Modulate CAG Repeat Expansion.

FEN1 cleavage activity on THF-containing CAG repeat flaps of different length. FEN1 substrates contained a 12-repeat (A) or 20-repeat CAG flap/hairpin (B), respectively. These substrates mimic BER intermediates generated by DNA strand slippage and pol β multinucleotide gap-filling synthesis on a THF-containing BER substrate. FEN1 endonucleolytic cleavage activity was examined as described under “Experimental Procedures” using 10 nm 5′-32P-labeled substrates. A schematic representation of the substrates is shown above the gels. A and B, Lane 1 correspond to incubation without enzyme. Lanes 2–6 correspond to reaction mixtures with 0.1, 0.5, 1, 5, and 10 nm FEN1, respectively. The configurations of 5′-THF-(CAG)12 (C) and 5′-THF-(CAG)20 (D) were probed by E. coli T5 exonuclease digestion under the conditions described under “Experimental Procedures.” The figure illustrates the sizes of the digestion products, and the fastest migrating band near the bottom of the gel corresponded to a 2-nucleotide fragment. C, lanes 2 and 3 represent digestion of (CAG)12-THF with 0.05 unit of and 0.1 unit of T5 exonuclease, respectively. D, lanes 2 and 3 represent digestion of (CAG)20-THF with 1 unit of and 5 units of T5 exonuclease, respectively. Specific digestion sites of T5 exonuclease on the (CAG)20-THF hairpin are illustrated schematically above the gel. Arrows indicate the main digestion products. In C and D, lane 1 represents the undigested substrate, and lane 4 represents markers (M) generated by DNase I digestion of the substrates.

Yuan Liu, et al. J Biol Chem. 2009 October 9;284(41):28352-28366.
6.
FIGURE 3.

FIGURE 3. From: Coordination between Polymerase ? and FEN1 Can Modulate CAG Repeat Expansion.

FEN1 stimulates CAG repeat expansion. The DNA substrate is illustrated schematically above the gel (in A, B, D, and E). A, the effect of FEN1 cleavage on CAG repeat expansion with THF-containing substrates. Substrates containing 19 and 24 CAG repeats were used. The reaction mixture contained 20 nm APE1, 2 nm pol β, 100 units of T4 DNA ligase, and 10 nm 5′-32P-labeled substrates with or without 5 nm FEN1 as indicated. The guanine of the first CAG of the substrates was replaced by a THF residue. Lanes 1 and 3 correspond to incubations in the absence of FEN1, whereas lanes 2 and 4 correspond to incubations in the presence of FEN1. B, the effect of FEN1 on CAG repeat expansion during repair of substrates with natural sugar phosphate (dRP). Incubations contained 20 nm UDG, 25 nm APE1, 2 nm pol β, and 100 units of T4 DNA ligase in the absence or presence of FEN1 (5 nm). 5′-32P-Labeled substrates (10 nm) containing 20 or 25 CAG repeats were used. The guanine of the first CAG of the substrate was replaced by a deoxyuridine. Lanes 1 and 2 show the reaction products formed in the absence and presence of FEN1, respectively. C, quantification of the amount of expansion products resulting from the experiments shown in A and B. The white bars illustrate the amount of expansion products generated in the absence of FEN1. The gray bars illustrate the amount of expansion products generated in the presence of FEN1. D, control experiments with random sequence substrates that contain THF and have the same length as either (CAG)20-THF (100 nt) or (CAG)25-THF (115 nt) were conducted under the conditions described in A. Unexpanded products (100 or 115 nt) are indicated by arrows. E, control experiments with random sequence substrates that contain a natural dRP and correspond to (CAG)20-dRP (100 nt) or (CAG)25-dRP (115 nt) were performed under the conditions described in B. Unexpanded products (100 or 115 nt) are indicated by arrows. F, the FEN1 effect on CAG repeat expansion was determined with the THF-containing (CAG)20 substrate in the absence of dNTPs under the conditions described in A. APE1 cleavage product is indicated by an arrow.

Yuan Liu, et al. J Biol Chem. 2009 October 9;284(41):28352-28366.
7.
SCHEME 1.

SCHEME 1. From: Coordination between Polymerase ? and FEN1 Can Modulate CAG Repeat Expansion.

Models illustrating CAG repeat stability modulated by coordination among BER enzymes and cofactors during LP-BER of 8-oxoG. BER of 8-oxoG in CAG repeats is initiated by OGG1 removal of the damaged base, leaving an abasic site or leaving a strand break with 5′-phosphate and 3′-blocked OH (not illustrated). APE1 incises the abasic site (5′), leaving a single nucleotide gap and a sugar phosphate flap. Repair of the single-stranded DNA break intermediate is subjected to alternative scenarios that could result in different consequences. If repair is conducted by SN-BER, where pol β fills the single nucleotide gap and removes the sugar phosphate flap with its dRP lyase activity, the repeat length will be maintained, i.e. without CAG expansion (not shown). The scheme illustrates LP-BER involving coordinated handoff of BER intermediates among pol β single nucleotide gap-filling FEN1 flap cleavage and DNA ligase that prevents CAG repeat expansion (left portion of Scheme 1, subpathway ). However, if this coordination is disrupted by spontaneous strand slippage and formation of hairpin loop structures during repair (right portion of Scheme 1), different consequences may occur, depending on repeat length. Short CAG repeats tend to adopt a flap configuration, as shown in subpathway . FEN1 can then remove the entire short repeat flap to prevent repeat expansion. Long repeats tend to form stable hairpins, as shown in subpathway . FEN1 cannot cleave at the 3′-base of the hairpin, and pol β is then forced to perform gap-filling synthesis to fill the multinucleotide gaps. The 5′-ends at the base of the hairpins then undergo realignment by reannealing to the template strand to form various sizes of CAG-containing hairpins with short 5′-flaps containing a sugar phosphate group. FEN1 is then forced to use its alternate cleavage activity to process the 5′-flap of these CAG-containing hairpins, leaving a ligatable nicked hairpin intermediate that is sealed by ligase, resulting in CAG repeat expansion. During this LP-BER, HMGB1 may facilitate CAG expansion by stimulating APE1 5′-abasic site incision activity and FEN1 alternate cleavage of short CAG-containing flaps with a sugar phosphate group at the 5′-end.

Yuan Liu, et al. J Biol Chem. 2009 October 9;284(41):28352-28366.
8.
FIGURE 1.

FIGURE 1. From: Coordination between Polymerase ? and FEN1 Can Modulate CAG Repeat Expansion.

DNA polymerase β promotes CAG repeat expansion during repair of 8-oxoG. A, oligonucleotide substrates with an 8-oxoG in the context of 20 CAG repeats or a random DNA sequence were preincubated with purified OGG1 and APE1, and experiments were conducted as described under “Experimental Procedures.” DNA substrates (25 nm) were then incubated with cell extract as indicated. Lanes 1–3 represent reaction mixtures containing extract from pol β wild-type cells (+/+), pol β null cells (−/−), and pol β null cells complemented with a high level expression of FLAG-tagged pol β (Comp). DNA substrates were 32P-radiolabeled at the 5′-end of the 8-oxoG-containing strand. A schematic representation of the substrates is shown above the gels. The expansion products and 100-nt unexpanded product are indicated by arrows. The sizes of the products are indicated by DNA size markers (M). B, the amounts of expansion products formed in extracts of wild-type cells (+/+), pol β null cells (−/−), and pol β null cells complemented with expression of pol β (Comp) were quantified as the intensity of radioactive signals. Expansion products with CAG repeat and random substrates are shown. C, the PCR-amplified 6-carboxyfluorescein-labeled DNA products at varying sizes were separated by capillary electrophoresis and are illustrated as peaks. The height of a peak indicates the abundance of the DNA product. The sizes of DNA products are illustrated in nucleotides. Molecular size markers and PCR products were run in parallel to calibrate size as indicated. D, the effect of pol β on CAG repeat expansion during repair of 8-oxoG was examined in the presence of 25 nm APE1, 5 nm FEN1, and 100 units of T4 DNA ligase as a function of increasing concentrations of pol β. 5′-32P-Labeled substrate (10 nm) containing 20 CAG repeats was utilized. Lane 1 represents a reaction mixture containing APE1 but without the other enzymes. The expansion products and unexpanded product are indicated by arrows. A schematic representation of the substrates is shown above the gels. E, the role of pol β in promoting CAG repeat expansion was further verified by complementing pol β null cell extract with purified pol β (right panel). The control experiment with random sequence substrate was performed under the same conditions (lanes 2–4, left panel). Lane 1 represents the reaction mixture with pol β null extract along. Lane 5 represents a mixture of three DNA markers of varying size as indicated (M).

Yuan Liu, et al. J Biol Chem. 2009 October 9;284(41):28352-28366.

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