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AP endonuclease 1 prevents trinucleotide repeat expansion via a novel mechanism during base excision repair.

Beaver JM, Lai Y, Xu M, Casin AH, Laverde EE, Liu Y - Nucleic Acids Res. (2015)

Bottom Line: Here, we further provide the first evidence that AP endonuclease 1 (APE1) prevented TNR expansions via its 3'-5' exonuclease activity and stimulatory effect on DNA ligation during BER in a hairpin loop.Coordinating with flap endonuclease 1, the APE1 3'-5' exonuclease activity cleaves the annealed upstream 3'-flap of a double-flap intermediate resulting from 5'-incision of an abasic site in the hairpin loop.Furthermore, APE1 stimulated DNA ligase I to resolve a long double-flap intermediate, thereby promoting hairpin removal and preventing TNR expansions.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA Biochemistry Ph.D. Program, Florida International University, Miami, FL 33199, USA.

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APE1 stimulates removal of a double-flap intermediate during BER. APE1 stimulation of resolution of the double-flap intermediate resulting from (CAG)7 and (CAG)14 hairpins during BER was examined by reconstituting BER with the substrates containing short (CAG)3/(CAG)4 double-flaps (left panel) or long (CAG)7/(CAG)7 double-flaps (right panel) with a THF residue (A) and without a THF residue (B) in the absence and presence of APE1. (A) Lanes 1 and 15 indicate the size markers of the template strand or unexpanded product. Lanes 2 and 16 represent the size markers of the damaged strand or expanded product. Lanes 3 and 17 represent the substrate only. Lanes 4–5 and lanes 18–19 correspond to the reaction mixture containing the substrates and 50 or 100 nM APE1. Lanes 6–11 and lanes 20–22 correspond to reactions with FEN1 (0.5 or 1 nM) and LIG I (5 nM) in the absence or presence of APE1. Lanes 12–14 and lanes 23–25 correspond to reactions with FEN1 (1 nM), LIG I (5 nM) and pol β (5 or 10 nM) in the absence and presence of APE1. (B) Lanes 1 and 18 indicate the size markers that represent the template strand or unexpanded product. Lanes 2 and 19 correspond to the size markers that illustrate the damaged strand. Lanes 3 and 20 correspond to the substrate only. Lanes 4–5 and lanes 21–22 correspond to the reaction mixture with the substrates and 50 or 100 nM APE1 only. Lanes 6–8 and lanes 23–25 correspond to reactions with the substrates and LIG I (5 nM) in the absence and presence of 50 or 100 nM APE1. Lanes 9–11 and lanes 26–28 correspond to reactions containing the substrates, pol β (10 nM) and LIG I (5 nM) in the absence and presence of APE1. Lanes 12–14 and lanes 29–31 correspond to reactions with the substrates, FEN1 (1 nM) and LIG I (5 nM) without or with APE1. Lanes 15–17 and lanes 32–34 correspond to reactions mixtures with the substrates, pol β (10 nM), FEN1 (1 nM) and LIG I (5 nM) without or with APE1.
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Figure 3: APE1 stimulates removal of a double-flap intermediate during BER. APE1 stimulation of resolution of the double-flap intermediate resulting from (CAG)7 and (CAG)14 hairpins during BER was examined by reconstituting BER with the substrates containing short (CAG)3/(CAG)4 double-flaps (left panel) or long (CAG)7/(CAG)7 double-flaps (right panel) with a THF residue (A) and without a THF residue (B) in the absence and presence of APE1. (A) Lanes 1 and 15 indicate the size markers of the template strand or unexpanded product. Lanes 2 and 16 represent the size markers of the damaged strand or expanded product. Lanes 3 and 17 represent the substrate only. Lanes 4–5 and lanes 18–19 correspond to the reaction mixture containing the substrates and 50 or 100 nM APE1. Lanes 6–11 and lanes 20–22 correspond to reactions with FEN1 (0.5 or 1 nM) and LIG I (5 nM) in the absence or presence of APE1. Lanes 12–14 and lanes 23–25 correspond to reactions with FEN1 (1 nM), LIG I (5 nM) and pol β (5 or 10 nM) in the absence and presence of APE1. (B) Lanes 1 and 18 indicate the size markers that represent the template strand or unexpanded product. Lanes 2 and 19 correspond to the size markers that illustrate the damaged strand. Lanes 3 and 20 correspond to the substrate only. Lanes 4–5 and lanes 21–22 correspond to the reaction mixture with the substrates and 50 or 100 nM APE1 only. Lanes 6–8 and lanes 23–25 correspond to reactions with the substrates and LIG I (5 nM) in the absence and presence of 50 or 100 nM APE1. Lanes 9–11 and lanes 26–28 correspond to reactions containing the substrates, pol β (10 nM) and LIG I (5 nM) in the absence and presence of APE1. Lanes 12–14 and lanes 29–31 correspond to reactions with the substrates, FEN1 (1 nM) and LIG I (5 nM) without or with APE1. Lanes 15–17 and lanes 32–34 correspond to reactions mixtures with the substrates, pol β (10 nM), FEN1 (1 nM) and LIG I (5 nM) without or with APE1.

Mentions: Our previous studies have shown that during BER of a base lesion within the loop of a CAG repeat hairpin, the hairpin is converted to a double-flap intermediate as a result of APE1 5′-incision of an abasic site (13). We found that the double-flap intermediate was then processed by a 3′-5′ flap endonuclease, Mus81/Eme1 and FEN1 5′-flap cleavage (13). This led to cleavage of the flaps, thereby resulting in the removal of the hairpin. We further hypothesized that APE1 is also involved in the processing of a double-flap intermediate by employing its 3′-5′ exonuclease activity to process the upstream 3′-flap, thereby promoting removal of a TNR hairpin structure. To test this, we reconstituted BER with a substrate containing an upstream 3′-(CAG)4 flap and a downstream 5′-(CAG)3 flap with a THF residue, as well as with a substrate containing a (CAG)7 flap on both the upstream and downstream strands with a 5′-THF residue, in the absence and presence of APE1. These substrates simulate the double-flap intermediates produced by APE1 5′-incision of the (CAG)7 and (CAG)14 hairpins, respectively. We found that BER reconstituted with FEN1 (0.5 and 1 nM) and LIG I (5 nM) without and with pol β (5 nM) on the (CAG)3/(CAG)4 double-flap substrate, resulted in the production of a significant amount of the repaired unexpanded product (Figure 3A, lanes 6, 9 and 12). However, 50 and 100 nM APE1 did not significantly alter the production of the unexpanded repaired product with the substrate (Figure 3A, compare lanes 7–8 with lane 6, lanes 10–11 with lane 9 and lanes 13–14 with lane 12). Further characterization of the 3′-5′ exonuclease activity of APE1 showed that 50 and 100 nM APE1 exhibited efficient 3′-5′ exonuclease activity that generated a significant amount of exonucleolytic cleavage products (Figure 3A, lanes 4–5). This indicated that APE1 cleaved the upstream CAG repeats exonucleolytically. The results showed that APE1 exonuclease did not play a significant role in the removal of the short double-flaps, suggesting that FEN1 flap cleavage plays a predominant role in processing the short double-flaps. Interestingly, we found that BER with the substrate containing long double-flaps in the presence of FEN1 (1 nM) and LIG I (5 nM) only resulted in a series of repaired products that are longer than the repaired unexpanded product (Figure 3A, lanes 20 and 23), but shorter than the original hairpin-containing substrate. They were termed ‘repaired expanded product’ (Figure 3A, lanes 20–25). This occurred in the absence and presence of pol β (10 nM) (Figure 3A, lanes 20–25) indicating that the production of the expanded products was independent of pol β. However, we found that the presence of 50 and 100 nM APE1 resulted in the formation of repaired unexpanded product (Figure 3A, lanes 21–22 and lanes 24–25). APE1 alone failed to generate the product (Figure 3A, lanes 18–19), indicating that the product was specifically generated through BER. However, APE1 failed to affect the production of repaired expansion products during BER (Figure 3A, compare the amount of repaired expanded products in lanes 21–22 and lanes 24–25 with that in lanes 20 and 23). This suggests that APE1 promoted the complete resolution of the long double-flap intermediate resulting from a (CAG)14 hairpin, thereby specifically stimulating the production of repaired unexpanded product. To further determine the effect that is specifically from APE1 on the production of repaired unexpanded products during BER, we reconstituted BER with 50 and 100 nM APE1 without or with a low concentration of FEN1 (1 nM) with the double-flap substrates without a THF residue. We found that on the short (CAG)3/(CAG)4 double-flap substrate, APE1 (50 and 100 nM) along with LIG I (5 nM) was sufficient to generate repaired unexpanded product in the absence and presence of 10 nM pol β (Figure 3B, lanes 7–8 and lanes 10–11). This indicated that APE1 facilitated the processing of the short (CAG)3/(CAG)4 double-flap by cleaving the upstream strand via its 3′-5′ exonuclease activity independent of FEN1. The production of the repaired unexpanded product was significantly stimulated by the presence of 1 nM FEN1 (Figure 3B, left panel, lanes 13–14 and lanes 16–17). To further determine the specific effect of APE1 on the removal of the long (CAG)7/(CAG)7 double-flaps, we reconstituted BER with the substrate containing (CAG)7/(CAG)7 double-flaps without the 5′-THF residue in the absence and presence of FEN1 (1 nM) (Figure 3B, right panel). We found that 50 and 100 nM APE1 alone or APE1 along with LIG I (5 nM) in the absence and presence of pol β (10 nM) failed to produce any repair products (Figure 3B, lanes 21–22, lanes 24–25 and lanes 27–28). BER reconstituted with FEN1 and LIG I produced only repaired expanded products without and with pol β (10 nM) (Figure 3B, lanes 29 and 32). However, BER reconstituted with APE1, FEN1 and LIG I in the absence and presence of pol β resulted in the production of a significant amount of the repaired unexpanded product (Figure 3B, lanes 30–31 and lanes 33–34). The results indicated that APE1 significantly promoted the removal of both the short (CAG)3/(CAG)4 and long (CAG)7/(CAG)7 double-flap intermediates. Thus, we conclude that APE1 can facilitate the resolution of the double-flap intermediate formed during BER in a CAG repeat hairpin loop and stimulate the formation of the unexpanded repaired product. APE1 alone can lead to complete removal of a short double-flap intermediate through its 3′-5′ exonuclease activity (Figure 3B, lanes 7–8). However, it can only promote the processing of an intermediate with long double-flaps by cooperating with FEN1 flap cleavage (Figure 3B, lanes 30–31 and lanes 33–34).


AP endonuclease 1 prevents trinucleotide repeat expansion via a novel mechanism during base excision repair.

Beaver JM, Lai Y, Xu M, Casin AH, Laverde EE, Liu Y - Nucleic Acids Res. (2015)

APE1 stimulates removal of a double-flap intermediate during BER. APE1 stimulation of resolution of the double-flap intermediate resulting from (CAG)7 and (CAG)14 hairpins during BER was examined by reconstituting BER with the substrates containing short (CAG)3/(CAG)4 double-flaps (left panel) or long (CAG)7/(CAG)7 double-flaps (right panel) with a THF residue (A) and without a THF residue (B) in the absence and presence of APE1. (A) Lanes 1 and 15 indicate the size markers of the template strand or unexpanded product. Lanes 2 and 16 represent the size markers of the damaged strand or expanded product. Lanes 3 and 17 represent the substrate only. Lanes 4–5 and lanes 18–19 correspond to the reaction mixture containing the substrates and 50 or 100 nM APE1. Lanes 6–11 and lanes 20–22 correspond to reactions with FEN1 (0.5 or 1 nM) and LIG I (5 nM) in the absence or presence of APE1. Lanes 12–14 and lanes 23–25 correspond to reactions with FEN1 (1 nM), LIG I (5 nM) and pol β (5 or 10 nM) in the absence and presence of APE1. (B) Lanes 1 and 18 indicate the size markers that represent the template strand or unexpanded product. Lanes 2 and 19 correspond to the size markers that illustrate the damaged strand. Lanes 3 and 20 correspond to the substrate only. Lanes 4–5 and lanes 21–22 correspond to the reaction mixture with the substrates and 50 or 100 nM APE1 only. Lanes 6–8 and lanes 23–25 correspond to reactions with the substrates and LIG I (5 nM) in the absence and presence of 50 or 100 nM APE1. Lanes 9–11 and lanes 26–28 correspond to reactions containing the substrates, pol β (10 nM) and LIG I (5 nM) in the absence and presence of APE1. Lanes 12–14 and lanes 29–31 correspond to reactions with the substrates, FEN1 (1 nM) and LIG I (5 nM) without or with APE1. Lanes 15–17 and lanes 32–34 correspond to reactions mixtures with the substrates, pol β (10 nM), FEN1 (1 nM) and LIG I (5 nM) without or with APE1.
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Figure 3: APE1 stimulates removal of a double-flap intermediate during BER. APE1 stimulation of resolution of the double-flap intermediate resulting from (CAG)7 and (CAG)14 hairpins during BER was examined by reconstituting BER with the substrates containing short (CAG)3/(CAG)4 double-flaps (left panel) or long (CAG)7/(CAG)7 double-flaps (right panel) with a THF residue (A) and without a THF residue (B) in the absence and presence of APE1. (A) Lanes 1 and 15 indicate the size markers of the template strand or unexpanded product. Lanes 2 and 16 represent the size markers of the damaged strand or expanded product. Lanes 3 and 17 represent the substrate only. Lanes 4–5 and lanes 18–19 correspond to the reaction mixture containing the substrates and 50 or 100 nM APE1. Lanes 6–11 and lanes 20–22 correspond to reactions with FEN1 (0.5 or 1 nM) and LIG I (5 nM) in the absence or presence of APE1. Lanes 12–14 and lanes 23–25 correspond to reactions with FEN1 (1 nM), LIG I (5 nM) and pol β (5 or 10 nM) in the absence and presence of APE1. (B) Lanes 1 and 18 indicate the size markers that represent the template strand or unexpanded product. Lanes 2 and 19 correspond to the size markers that illustrate the damaged strand. Lanes 3 and 20 correspond to the substrate only. Lanes 4–5 and lanes 21–22 correspond to the reaction mixture with the substrates and 50 or 100 nM APE1 only. Lanes 6–8 and lanes 23–25 correspond to reactions with the substrates and LIG I (5 nM) in the absence and presence of 50 or 100 nM APE1. Lanes 9–11 and lanes 26–28 correspond to reactions containing the substrates, pol β (10 nM) and LIG I (5 nM) in the absence and presence of APE1. Lanes 12–14 and lanes 29–31 correspond to reactions with the substrates, FEN1 (1 nM) and LIG I (5 nM) without or with APE1. Lanes 15–17 and lanes 32–34 correspond to reactions mixtures with the substrates, pol β (10 nM), FEN1 (1 nM) and LIG I (5 nM) without or with APE1.
Mentions: Our previous studies have shown that during BER of a base lesion within the loop of a CAG repeat hairpin, the hairpin is converted to a double-flap intermediate as a result of APE1 5′-incision of an abasic site (13). We found that the double-flap intermediate was then processed by a 3′-5′ flap endonuclease, Mus81/Eme1 and FEN1 5′-flap cleavage (13). This led to cleavage of the flaps, thereby resulting in the removal of the hairpin. We further hypothesized that APE1 is also involved in the processing of a double-flap intermediate by employing its 3′-5′ exonuclease activity to process the upstream 3′-flap, thereby promoting removal of a TNR hairpin structure. To test this, we reconstituted BER with a substrate containing an upstream 3′-(CAG)4 flap and a downstream 5′-(CAG)3 flap with a THF residue, as well as with a substrate containing a (CAG)7 flap on both the upstream and downstream strands with a 5′-THF residue, in the absence and presence of APE1. These substrates simulate the double-flap intermediates produced by APE1 5′-incision of the (CAG)7 and (CAG)14 hairpins, respectively. We found that BER reconstituted with FEN1 (0.5 and 1 nM) and LIG I (5 nM) without and with pol β (5 nM) on the (CAG)3/(CAG)4 double-flap substrate, resulted in the production of a significant amount of the repaired unexpanded product (Figure 3A, lanes 6, 9 and 12). However, 50 and 100 nM APE1 did not significantly alter the production of the unexpanded repaired product with the substrate (Figure 3A, compare lanes 7–8 with lane 6, lanes 10–11 with lane 9 and lanes 13–14 with lane 12). Further characterization of the 3′-5′ exonuclease activity of APE1 showed that 50 and 100 nM APE1 exhibited efficient 3′-5′ exonuclease activity that generated a significant amount of exonucleolytic cleavage products (Figure 3A, lanes 4–5). This indicated that APE1 cleaved the upstream CAG repeats exonucleolytically. The results showed that APE1 exonuclease did not play a significant role in the removal of the short double-flaps, suggesting that FEN1 flap cleavage plays a predominant role in processing the short double-flaps. Interestingly, we found that BER with the substrate containing long double-flaps in the presence of FEN1 (1 nM) and LIG I (5 nM) only resulted in a series of repaired products that are longer than the repaired unexpanded product (Figure 3A, lanes 20 and 23), but shorter than the original hairpin-containing substrate. They were termed ‘repaired expanded product’ (Figure 3A, lanes 20–25). This occurred in the absence and presence of pol β (10 nM) (Figure 3A, lanes 20–25) indicating that the production of the expanded products was independent of pol β. However, we found that the presence of 50 and 100 nM APE1 resulted in the formation of repaired unexpanded product (Figure 3A, lanes 21–22 and lanes 24–25). APE1 alone failed to generate the product (Figure 3A, lanes 18–19), indicating that the product was specifically generated through BER. However, APE1 failed to affect the production of repaired expansion products during BER (Figure 3A, compare the amount of repaired expanded products in lanes 21–22 and lanes 24–25 with that in lanes 20 and 23). This suggests that APE1 promoted the complete resolution of the long double-flap intermediate resulting from a (CAG)14 hairpin, thereby specifically stimulating the production of repaired unexpanded product. To further determine the effect that is specifically from APE1 on the production of repaired unexpanded products during BER, we reconstituted BER with 50 and 100 nM APE1 without or with a low concentration of FEN1 (1 nM) with the double-flap substrates without a THF residue. We found that on the short (CAG)3/(CAG)4 double-flap substrate, APE1 (50 and 100 nM) along with LIG I (5 nM) was sufficient to generate repaired unexpanded product in the absence and presence of 10 nM pol β (Figure 3B, lanes 7–8 and lanes 10–11). This indicated that APE1 facilitated the processing of the short (CAG)3/(CAG)4 double-flap by cleaving the upstream strand via its 3′-5′ exonuclease activity independent of FEN1. The production of the repaired unexpanded product was significantly stimulated by the presence of 1 nM FEN1 (Figure 3B, left panel, lanes 13–14 and lanes 16–17). To further determine the specific effect of APE1 on the removal of the long (CAG)7/(CAG)7 double-flaps, we reconstituted BER with the substrate containing (CAG)7/(CAG)7 double-flaps without the 5′-THF residue in the absence and presence of FEN1 (1 nM) (Figure 3B, right panel). We found that 50 and 100 nM APE1 alone or APE1 along with LIG I (5 nM) in the absence and presence of pol β (10 nM) failed to produce any repair products (Figure 3B, lanes 21–22, lanes 24–25 and lanes 27–28). BER reconstituted with FEN1 and LIG I produced only repaired expanded products without and with pol β (10 nM) (Figure 3B, lanes 29 and 32). However, BER reconstituted with APE1, FEN1 and LIG I in the absence and presence of pol β resulted in the production of a significant amount of the repaired unexpanded product (Figure 3B, lanes 30–31 and lanes 33–34). The results indicated that APE1 significantly promoted the removal of both the short (CAG)3/(CAG)4 and long (CAG)7/(CAG)7 double-flap intermediates. Thus, we conclude that APE1 can facilitate the resolution of the double-flap intermediate formed during BER in a CAG repeat hairpin loop and stimulate the formation of the unexpanded repaired product. APE1 alone can lead to complete removal of a short double-flap intermediate through its 3′-5′ exonuclease activity (Figure 3B, lanes 7–8). However, it can only promote the processing of an intermediate with long double-flaps by cooperating with FEN1 flap cleavage (Figure 3B, lanes 30–31 and lanes 33–34).

Bottom Line: Here, we further provide the first evidence that AP endonuclease 1 (APE1) prevented TNR expansions via its 3'-5' exonuclease activity and stimulatory effect on DNA ligation during BER in a hairpin loop.Coordinating with flap endonuclease 1, the APE1 3'-5' exonuclease activity cleaves the annealed upstream 3'-flap of a double-flap intermediate resulting from 5'-incision of an abasic site in the hairpin loop.Furthermore, APE1 stimulated DNA ligase I to resolve a long double-flap intermediate, thereby promoting hairpin removal and preventing TNR expansions.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA Biochemistry Ph.D. Program, Florida International University, Miami, FL 33199, USA.

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Related in: MedlinePlus