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Figure 6. The DNA lyase activity of hNTHL1 contributes to the processing of lesions in nucleosomes, even in the ... Figure 6. The DNA lyase activity of.

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Presentation on theme: "Figure 6. The DNA lyase activity of hNTHL1 contributes to the processing of lesions in nucleosomes, even in the ... Figure 6. The DNA lyase activity of."— Presentation transcript:

1 Figure 6. The DNA lyase activity of hNTHL1 contributes to the processing of lesions in nucleosomes, even in the ... Figure 6. The DNA lyase activity of hNTHL1 contributes to the processing of lesions in nucleosomes, even in the presence of APE1. To distinguish between APE1- and hNTHL1-mediated strand incision activities, Tg-containing DNA and nucleosomal substrates were labeled with <sup>32</sup>P at a position 3′ to the lesion site (see Figure 1) nM of the resulting substrates were incubated with 2 nM NTHL1, in the presence or absence of 0.2 nM APE1. Reactions were halted after 5 min, and samples were processed and reaction products fractionated and analyzed as described in the Methods. The Figure shows products separated on 20% denaturing polyacrylamide gels, Naked DNA lanes contained 20% as much sample as the nucleosomal DNA lanes equalize relative intensities of the product bands to aid comparison of band migration. Product bands resulting from either APE1 activity or NTHL1 activity are indicated (see also Figure 1B). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Res, Volume 47, Issue 6, 16 January 2019, Pages 2922–2931, The content of this slide may be subject to copyright: please see the slide notes for details.

2 Figure 5. The processing of DNA intermediates generated during the hNTHL1-initiated repair of Tg residues in ... Figure 5. The processing of DNA intermediates generated during the hNTHL1-initiated repair of Tg residues in nucleosomes is enhanced to a greater extent by increasing APE1 concentrations in reactions with outward-facing Tg lesions than for inward-facing Tg lesions. Reactions contained 2.25 nM Tg substrate, 2 nM NTHL1, and either 0.2, 2.0 or 20 nM APE1. Aliquots were taken at various time points and analyzed as before. The abundance of intermediates, as a fraction of total product, was graphed as a function of time. Error bars represent standard deviation of three independent experiments. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Res, Volume 47, Issue 6, 16 January 2019, Pages 2922–2931, The content of this slide may be subject to copyright: please see the slide notes for details.

3 Figure 4. More efficient processing of DNA intermediates by APE1 during the repair of Tg-containing nucleosomes in ... Figure 4. More efficient processing of DNA intermediates by APE1 during the repair of Tg-containing nucleosomes in reactions containing a Δ63 truncation mutant of hNTHL1 instead of full-length hNTHL1. Graph showing the abundance of intermediates as a fraction of total product, as a function of time, in reactions containing 2.25 nM Tg substrate, 0.2 nM APE1 and 2 nM Δ63 hNTHL1 N-terminal truncation (dotted line traces). For ease of comparison, the same graph shows data from Figure 3B for reactions with full length NTHL1 (solid line traces). Error bars represent standard deviation of three independent experiments. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Res, Volume 47, Issue 6, 16 January 2019, Pages 2922–2931, The content of this slide may be subject to copyright: please see the slide notes for details.

4 Figure 3. DNA intermediates generated during the hNTHL1-initiated repair of Tg-containing nucleosomes accumulate to a ... Figure 3. DNA intermediates generated during the hNTHL1-initiated repair of Tg-containing nucleosomes accumulate to a greater extent than in reactions with naked DNA substrates. (A) A representative gel showing the various products produced in reactions containing 2.25 nM Tg substrate, in the naked or nucleosomal forms and 2 nM hNTHL1, in the presence and absence of 0.2 nM APE1. (B) Graph showing the abundance of intermediates as a fraction of total product, as a function of time for reactions containing 2.25 nM Tg substrate, in the naked or nucleosomal forms with 2 nM hNTHL1 and 0.2 nM APE1. Error bars represent standard deviation of three independent experiments. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Res, Volume 47, Issue 6, 16 January 2019, Pages 2922–2931, The content of this slide may be subject to copyright: please see the slide notes for details.

5 Figure 2. APE1 stimulates the hNTHL1-mediated excision of oxidized bases from naked DNA but not from nucleosomes. ... Figure 2. APE1 stimulates the hNTHL1-mediated excision of oxidized bases from naked DNA but not from nucleosomes. Nucleosomes containing inward (yellow)- or outward (blue)-facing Tg residues, and their respective naked DNA controls (inward green, outward pink), were incubated with hNTHL1, in the presence or absence of APE1. Reactions contained 2.25 nM Tg substrate and 0.2 nM (A and B) or 2 nM hNTHL1 (C and D). Reactions that also contained 0.2 nM APE1 are indicated with a dotted line trace and control reaction with no APE1 are indicated with solid traces. Aliquots taken at the times indicated were processed, and reaction products visualized and quantified, as described in the Methods. Error bars represent standard deviation of three independent experiments. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Res, Volume 47, Issue 6, 16 January 2019, Pages 2922–2931, The content of this slide may be subject to copyright: please see the slide notes for details.

6 Figure 1. (A) The 5S rDNA sequence from L
Figure 1. (A) The 5S rDNA sequence from L. variegatus used to generate nucleosomes with discretely positioned ... Figure 1. (A) The 5S rDNA sequence from L. variegatus used to generate nucleosomes with discretely positioned lesions. (B) Enzymatic steps during base excision repair (BER). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Res, Volume 47, Issue 6, 16 January 2019, Pages 2922–2931, The content of this slide may be subject to copyright: please see the slide notes for details.

7 Figure 7. Tetrahydrofuran and polyunsaturated aldehyde (PUA)-containing DNA’s are equally robust substrates for APE1 ... Figure 7. Tetrahydrofuran and polyunsaturated aldehyde (PUA)-containing DNA’s are equally robust substrates for APE1 when packaged in nucleosomes. Reactions contained 2.25 nM substrate containing either a PUA (open symbols) or tetrahydrofuran (closed symbols) residue, and 0.2, 2 or 10 nM APE1. Solid line traces indicate data from DNA reactions and dotted line traces indicate data from nucleosome reactions. Reactions were quenched and samples processed and analyzed, as described in the Methods. Error bars represent standard deviation of three independent experiments. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Res, Volume 47, Issue 6, 16 January 2019, Pages 2922–2931, The content of this slide may be subject to copyright: please see the slide notes for details.

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