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. 2014 Jan 14;42(6):3675–3691. doi: 10.1093/nar/gkt1372

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© The Author(s) 2014. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — (A) Processing of a CAG repeat double-flap intermediate during BER prevented or attenuated CAG repeat expansion. Processing of a CAG repeat double-flap substrate and its effects on repeat stability during BER was examined with substrates containing an upstream 3′-(CAG)₄ and a downstream 5′-THF-(CAG)₃ (left panel) or an upstream 3′-(CAG)₇ flap and a downstream 5′-THF-(CAG)₇ flap (right panel). Lanes 1 and 11 represent substrates only. Lanes 2, 4, 12 and 14 correspond to reaction mixtures with or without 5 nM pol β in the presence of 10 nM FEN1 and 5 nM LIG I. Lanes 3, 5, 13 and 15 correspond reaction mixtures with or without 5 nM pol β in the presence of 250 nM Mus81/Eme1, 10 nM FEN1 and 5 nM LIG I. Lanes 6–7 and 16–17 represent reaction mixtures with or without 5 nM pol β in the presence of 250 nM Mus81/Eme1. Lanes 8–9 and 18–19 correspond to reaction mixtures with or without 5 nM pol β in the presence of 250 nM Mus81/Eme1 and 10 nM FEN1. Lanes 10 and 20 correspond to synthesized size markers (M). Substrates were ³²P-labeled at the 5′-end of their upstream strands. (B) Mus81/Eme1 cleavage of the upstream flap of a CAG repeat double-flap intermediate was examined using a double-flap substrate with an upstream 3′-(CAG)₄ and a downstream 5′-(CAG)₃-THF flap (left panel) or a substrate with an upstream 3′-(CAG)₇ flap and a downstream 5′-(CAG)₇-THF flap (right panel). The substrates were radiolabeled at the 3′-end of the upstream strand. Lanes 1 and 7 represent a substrate alone. Lanes 2 and 8 represent reaction mixtures with 250 nM Mus81/Eme1. Lanes 3 and 9 correspond to reaction mixtures with 250 nM Mus81/Eme1 and 5 nM pol β. Lanes 4 and 10 represent reaction mixtures with 250 nM Mus81/Eme1 and 10 nM FEN1. Lanes 5 and 11 correspond to reaction mixtures with 250 nM Mus81/Eme1, 5 nM pol β and 10 nM FEN1. Lanes 6 and 12 represent synthesized size markers (M). The substrates are illustrated schematically above the gels. A scheme that indicates Mus81/Eme1 cleavage of the upstream 3′-flap of the double-flap substrates is illustrated below the gels.

Figure 3. — (A) Processing of a CAG repeat double-flap intermediate during BER prevented or attenuated CAG repeat expansion. Processing of a CAG repeat double-flap substrate and its effects on repeat stability during BER was examined with substrates containing an upstream 3′-(CAG)₄ and a downstream 5′-THF-(CAG)₃ (left panel) or an upstream 3′-(CAG)₇ flap and a downstream 5′-THF-(CAG)₇ flap (right panel). Lanes 1 and 11 represent substrates only. Lanes 2, 4, 12 and 14 correspond to reaction mixtures with or without 5 nM pol β in the presence of 10 nM FEN1 and 5 nM LIG I. Lanes 3, 5, 13 and 15 correspond reaction mixtures with or without 5 nM pol β in the presence of 250 nM Mus81/Eme1, 10 nM FEN1 and 5 nM LIG I. Lanes 6–7 and 16–17 represent reaction mixtures with or without 5 nM pol β in the presence of 250 nM Mus81/Eme1. Lanes 8–9 and 18–19 correspond to reaction mixtures with or without 5 nM pol β in the presence of 250 nM Mus81/Eme1 and 10 nM FEN1. Lanes 10 and 20 correspond to synthesized size markers (M). Substrates were ³²P-labeled at the 5′-end of their upstream strands. (B) Mus81/Eme1 cleavage of the upstream flap of a CAG repeat double-flap intermediate was examined using a double-flap substrate with an upstream 3′-(CAG)₄ and a downstream 5′-(CAG)₃-THF flap (left panel) or a substrate with an upstream 3′-(CAG)₇ flap and a downstream 5′-(CAG)₇-THF flap (right panel). The substrates were radiolabeled at the 3′-end of the upstream strand. Lanes 1 and 7 represent a substrate alone. Lanes 2 and 8 represent reaction mixtures with 250 nM Mus81/Eme1. Lanes 3 and 9 correspond to reaction mixtures with 250 nM Mus81/Eme1 and 5 nM pol β. Lanes 4 and 10 represent reaction mixtures with 250 nM Mus81/Eme1 and 10 nM FEN1. Lanes 5 and 11 correspond to reaction mixtures with 250 nM Mus81/Eme1, 5 nM pol β and 10 nM FEN1. Lanes 6 and 12 represent synthesized size markers (M). The substrates are illustrated schematically above the gels. A scheme that indicates Mus81/Eme1 cleavage of the upstream 3′-flap of the double-flap substrates is illustrated below the gels.