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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 LIG I activity on a double-flap intermediate. The effects of APE1 to stimulate the ligation activity of LIG I on the short (CAG)3/(CAG)4 and long (CAG)7/(CAG)7 double-flap intermediates during BER were determined by reconstituting BER in the absence and presence of low concentrations of APE1 (0.5 and 1 nM) at which a little or no 3′-5′ exonuclease activity was observed (Supplementary Figure S1) on the short and long double-flap substrates, respectively. Lanes 1 and 10 indicate the size markers of the template strand, corresponding to complete removal of the double-flaps and full repair. Lanes 2 and 11 correspond to the markers that illustrate the length of the damaged strand containing a CAG repeat hairpin and the size of repaired expanded product. Lanes 3 and 12 correspond to the substrate only. Lanes 4–6 and lanes 13–15 correspond to reactions containing the substrates, FEN1 (5 nM) and LIG I (0.1 nM or 1 nM) in the absence and presence of 0.5 or 1 nM APE1. Lanes 7–9 and lanes 16–18 correspond to reaction mixture containing the substrates, FEN1 (5 nM), LIG I (0.1 nM or 1 nM) and pol β (5 nM) in the absence and presence of 0.5 or 1 nM APE1. Substrates were 32P-labeled at the 5′-end of the upstream strands and are illustrated above each gel.
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Figure 6: APE1 stimulates LIG I activity on a double-flap intermediate. The effects of APE1 to stimulate the ligation activity of LIG I on the short (CAG)3/(CAG)4 and long (CAG)7/(CAG)7 double-flap intermediates during BER were determined by reconstituting BER in the absence and presence of low concentrations of APE1 (0.5 and 1 nM) at which a little or no 3′-5′ exonuclease activity was observed (Supplementary Figure S1) on the short and long double-flap substrates, respectively. Lanes 1 and 10 indicate the size markers of the template strand, corresponding to complete removal of the double-flaps and full repair. Lanes 2 and 11 correspond to the markers that illustrate the length of the damaged strand containing a CAG repeat hairpin and the size of repaired expanded product. Lanes 3 and 12 correspond to the substrate only. Lanes 4–6 and lanes 13–15 correspond to reactions containing the substrates, FEN1 (5 nM) and LIG I (0.1 nM or 1 nM) in the absence and presence of 0.5 or 1 nM APE1. Lanes 7–9 and lanes 16–18 correspond to reaction mixture containing the substrates, FEN1 (5 nM), LIG I (0.1 nM or 1 nM) and pol β (5 nM) in the absence and presence of 0.5 or 1 nM APE1. Substrates were 32P-labeled at the 5′-end of the upstream strands and are illustrated above each gel.

Mentions: A study from the Bambara group showed that APE1 can stimulate LIG I activity to promote ligation of a nick and facilitate the formation of repaired products during BER (25). Thus, it is possible that APE1 may also stimulate LIG I activity to promote the removal of a CAG repeat hairpin and facilitate the production of the repaired products during BER of a base lesion in a hairpin loop. To test this possibility, we determined the effects of APE1 on LIG I activity independent of its 3′-5′ exonuclease activity by measuring the production of the repaired products in the presence of low concentrations of APE1 (0.5 and 1 nM) that exhibited a low 3′-5′ exonuclease activity on the (CAG)3/(CAG)4 double-flap substrate (Supplementary Figure S1, lanes 3–4) and little exonucleolytic activity on the (CAG)7/(CAG)7 double-flap substrate (Supplementary Figure S1, lanes 9–10). We found that low concentrations of APE1 failed to alter the production of repaired products resulting from a short double-flap substrate (Figure 6, compare lanes 5–6 with lane 4 and lanes 8–9 with lane 7) indicating that APE1 did not significantly alter LIG I activity during removal of a small hairpin. However, the same concentrations of APE1 resulted in the production of the unexpanded product on the long (CAG)7/(CAG)7 double-flap substrate (Figure 6, compare lanes 14–15 with lane 13 and lanes 17–18 with lane 16). The results indicated that APE1 stimulated the ligation activity of LIG I, thereby promoting the production of the unexpanded repair product. Interestingly, we observed that the same concentrations of APE1 failed to stimulate the production of shortened repaired expanded products (Figure 6, lanes 14–15, lanes 17–18) suggesting that APE1 failed to stimulate the ligation of a nick by LIG I that was adjacent a hairpin structure.


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 LIG I activity on a double-flap intermediate. The effects of APE1 to stimulate the ligation activity of LIG I on the short (CAG)3/(CAG)4 and long (CAG)7/(CAG)7 double-flap intermediates during BER were determined by reconstituting BER in the absence and presence of low concentrations of APE1 (0.5 and 1 nM) at which a little or no 3′-5′ exonuclease activity was observed (Supplementary Figure S1) on the short and long double-flap substrates, respectively. Lanes 1 and 10 indicate the size markers of the template strand, corresponding to complete removal of the double-flaps and full repair. Lanes 2 and 11 correspond to the markers that illustrate the length of the damaged strand containing a CAG repeat hairpin and the size of repaired expanded product. Lanes 3 and 12 correspond to the substrate only. Lanes 4–6 and lanes 13–15 correspond to reactions containing the substrates, FEN1 (5 nM) and LIG I (0.1 nM or 1 nM) in the absence and presence of 0.5 or 1 nM APE1. Lanes 7–9 and lanes 16–18 correspond to reaction mixture containing the substrates, FEN1 (5 nM), LIG I (0.1 nM or 1 nM) and pol β (5 nM) in the absence and presence of 0.5 or 1 nM APE1. Substrates were 32P-labeled at the 5′-end of the upstream strands and are illustrated above each gel.
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Figure 6: APE1 stimulates LIG I activity on a double-flap intermediate. The effects of APE1 to stimulate the ligation activity of LIG I on the short (CAG)3/(CAG)4 and long (CAG)7/(CAG)7 double-flap intermediates during BER were determined by reconstituting BER in the absence and presence of low concentrations of APE1 (0.5 and 1 nM) at which a little or no 3′-5′ exonuclease activity was observed (Supplementary Figure S1) on the short and long double-flap substrates, respectively. Lanes 1 and 10 indicate the size markers of the template strand, corresponding to complete removal of the double-flaps and full repair. Lanes 2 and 11 correspond to the markers that illustrate the length of the damaged strand containing a CAG repeat hairpin and the size of repaired expanded product. Lanes 3 and 12 correspond to the substrate only. Lanes 4–6 and lanes 13–15 correspond to reactions containing the substrates, FEN1 (5 nM) and LIG I (0.1 nM or 1 nM) in the absence and presence of 0.5 or 1 nM APE1. Lanes 7–9 and lanes 16–18 correspond to reaction mixture containing the substrates, FEN1 (5 nM), LIG I (0.1 nM or 1 nM) and pol β (5 nM) in the absence and presence of 0.5 or 1 nM APE1. Substrates were 32P-labeled at the 5′-end of the upstream strands and are illustrated above each gel.
Mentions: A study from the Bambara group showed that APE1 can stimulate LIG I activity to promote ligation of a nick and facilitate the formation of repaired products during BER (25). Thus, it is possible that APE1 may also stimulate LIG I activity to promote the removal of a CAG repeat hairpin and facilitate the production of the repaired products during BER of a base lesion in a hairpin loop. To test this possibility, we determined the effects of APE1 on LIG I activity independent of its 3′-5′ exonuclease activity by measuring the production of the repaired products in the presence of low concentrations of APE1 (0.5 and 1 nM) that exhibited a low 3′-5′ exonuclease activity on the (CAG)3/(CAG)4 double-flap substrate (Supplementary Figure S1, lanes 3–4) and little exonucleolytic activity on the (CAG)7/(CAG)7 double-flap substrate (Supplementary Figure S1, lanes 9–10). We found that low concentrations of APE1 failed to alter the production of repaired products resulting from a short double-flap substrate (Figure 6, compare lanes 5–6 with lane 4 and lanes 8–9 with lane 7) indicating that APE1 did not significantly alter LIG I activity during removal of a small hairpin. However, the same concentrations of APE1 resulted in the production of the unexpanded product on the long (CAG)7/(CAG)7 double-flap substrate (Figure 6, compare lanes 14–15 with lane 13 and lanes 17–18 with lane 16). The results indicated that APE1 stimulated the ligation activity of LIG I, thereby promoting the production of the unexpanded repair product. Interestingly, we observed that the same concentrations of APE1 failed to stimulate the production of shortened repaired expanded products (Figure 6, lanes 14–15, lanes 17–18) suggesting that APE1 failed to stimulate the ligation of a nick by LIG I that was adjacent a hairpin structure.

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.

Show MeSH
Related in: MedlinePlus