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<input type="email" aria-label="Sender Email Address" placeholder="email@example.com" id="email-from" class="email-from" pattern="^[a-zA-Z0-9_.+-]+@[a-zA-Z0-9-]+\.[a-zA-Z0-9-.]+$" maxlength="256">
</div>
<div class="action-panel-control-wrap">
<label for="email-citation-format" class="action-panel-label">
Format:
</label>
<select id="email-citation-format" name="citation-format" class="action-panel-selector email-citation-format">
<option selected="selected" value="summary">Summary</option>
<option value="summary-text">Summary (text)</option>
<option value="abstract">Abstract</option>
<option value="abstract-text">Abstract (text)</option>
</select>
</div>
<div class="include-supplemental-container">
<input type="checkbox" aria-label="Include MeSH and other data" name="include-supplemental" id="email-include-supplemental" class="email-include-supplemental">
<label for="email-include-supplemental" class="email-include-supplemental-label">MeSH and other data</label>
</div>
<div class="form-field recaptcha ">
<div class="g-recaptcha" id="id-recaptcha" data-sitekey="6LfsWHMdAAAAAClKbtOpjQ2pMjgsGxvv7NdZW9uI"></div>
</div>
<div id="captcha-error-message" class="usa-input-error-message captcha-validation-message" role="alert"></div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="send">
Send email
</button>
<button class="action-panel-cancel"
aria-label="Close 'Email citations' panel"
ref="linksrc=close_email_panel"
aria-controls="email-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="cancel">
Cancel
</button>
</div>
<input type="hidden" name="email-search-details" value="" />
<input type="hidden" name="email-search-details-hash" value="0e42663a6c3bd85498fcb88798998fed7bfdc45d457db35281e41afe13cc0524" />
</form>
</div>
</div>
<div id="collections-action-panel"
class="collections-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-collections-open-panel-enabled="false"
data-collections-open-panel-url-hash="#open-collections-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to Collections
</h3>
<form id="collections-action-panel-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-myncbi-max-collection-name-length="100"
data-add-to-collection-max-amount="1000"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/">
<input type="hidden" name="csrfmiddlewaretoken" value="MBpyoDO79q6YbqK9nit6f2fg9drTisTL3o9D1thtJ1Ts5RQZ0B68JaLUHh95b3r5">
<div class="choice-group" role="radiogroup">
<ul class="radio-group-items">
<li>
<input type="radio"
id="collections-action-panel-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_new">
<label for="collections-action-panel-new">Create a new collection</label>
</li>
<li>
<input type="radio"
id="collections-action-panel-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_existing">
<label for="collections-action-panel-existing">Add to an existing collection</label>
</li>
</ul>
</div>
<div class="controls-wrapper">
<div class="action-panel-control-wrap new-collections-controls">
<label for="collections-action-panel-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label>
<input
type="text"
name="add-to-new-collection"
id="collections-action-panel-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="save_share"
data-ga-action="create_collection"
data-ga-label="non_favorties_collection">
<div class="collections-new-name-too-long usa-input-error-message selection-validation-message">
Name must be less than 100 characters
</div>
</div>
<div class="action-panel-control-wrap existing-collections-controls">
<label for="collections-action-panel-add-to-existing" class="action-panel-label">
Choose a collection:
</label>
<select id="collections-action-panel-add-to-existing"
class="action-panel-selector collections-action-add-to-existing"
data-ga-category="save_share"
data-ga-action="select_collection"
data-ga-label="($('#collections-action-add-to-existing').val() === 'Favorites') ? 'Favorites' : 'non_favorites_collection'">
</select>
<div class="collections-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your collection due to an error<br>
<a href="#">Please try again</a>
</div>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="add">
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to Collections' panel"
ref="linksrc=close_collections_panel"
aria-controls="collections-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="bibliography-action-panel"
class="bibliography-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-bibliography-open-panel-enabled="false"
data-bibliography-open-panel-url-hash="#open-bibliography-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to My Bibliography
</h3>
<form id="bibliography-action-panel-form"
class="bibliography-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-add-to-bibliography-max-amount="100"
data-add-to-bibliography-batch-size="10"
data-bibliography-delegates-url="/list-bibliography-delegates/"
data-add-to-bibliography-url="/add-to-bibliography/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-mybib-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/mybibliography/">
<input type="hidden" name="csrfmiddlewaretoken" value="MBpyoDO79q6YbqK9nit6f2fg9drTisTL3o9D1thtJ1Ts5RQZ0B68JaLUHh95b3r5">
<div class="action-panel-control-wrap bibliographies-controls">
<div class="choice-group">
<ul class="bibliographies-action-add radio-group-items">
<li>
<input name="bibliography" id="my-bibliography" class="my-bibliography" type="radio" checked/>
<label for="my-bibliography">My Bibliography</label>
</li>
</ul>
</div>
</div>
<div class="bibliographies-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your delegates due to an error<br>
<a href="#">Please try again</a>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore>
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to bibliography' panel"
ref="linksrc=close_bibliography_panel"
aria-controls="bibliography-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="mybib"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="saved-search-action-panel" class="saved-search-action-panel action-panel " aria-hidden="true"
data-saved-search-open-panel-enabled="false"
data-saved-search-open-panel-url-hash="#open-saved-search-panel">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your saved search
</h2>
<form id="saved-search-action-panel-form"
class="saved-search-action-panel-form action-panel-content action-form"
data-create-saved-search-url="/create-saved-search/"
data-try-search-terms-url="/try-search-term/"
data-saved-search-root-url="https://www.ncbi.nlm.nih.gov/myncbi/searches/">
<input type="hidden" name="csrfmiddlewaretoken" value="MBpyoDO79q6YbqK9nit6f2fg9drTisTL3o9D1thtJ1Ts5RQZ0B68JaLUHh95b3r5">
<div class="action-panel-control-wrap">
<label for="saved-search-name" class="action-panel-label saved-search-name-label required-field-asterisk">
Name of saved search:
</label>
<input maxlength="200"
type="text"
name="saved-search-name"
id="saved-search-name"
class="saved-search-name"
value=""
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-term" class="action-panel-label required-field-asterisk">
Search terms:
</label>
<textarea name="saved-search-term" id="saved-search-term" class="saved-search-term" required></textarea>
</div>
<div class="test-search-term-wrap">
<a href="#" class="try-search-term">Test search terms</a>
</div>
<div class="choice-group action-panel-extra-margin-top">
<span class="action-panel-label" id="fieldset-label">
Would you like email updates of new search results?
</span>
<fieldset id="saved-search-alert" aria-describedby="fieldset-label">
<legend class="usa-sr-only">Saved Search Alert Radio Buttons</legend>
<ul class="radio-group-items">
<li>
<input type="radio" id="saved-search-alert-yes" class="saved-search-alert-yes" name="saved-search-alert" value="yes" checked>
<label for="saved-search-alert-yes" class="action-panel-label">Yes</label>
</li>
<li>
<input aria-label="No radio input" type="radio" id="saved-search-alert-no" class="saved-search-alert-no" name="saved-search-alert" value="no">
<label for="saved-search-alert-no" class="action-panel-label">No</label>
</li>
</ul>
</fieldset>
</div>
<div class="alert-schedule-wrap">
<div class="action-panel-control-wrap">
<label class="action-panel-label">
Email:
</label>
<span aria-label="Email address" id="saved-search-email" class="action-panel-label"><span class="action-panel-label-bold"></span> (<a class="myncbi-account-settings" href="https://www.ncbi.nlm.nih.gov/account/settings/">change</a>)</span>
</div>
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="saved-search-frequency" class="action-panel-label">
Frequency:
</label>
<select id="saved-search-frequency" class="no-border-panel-selector saved-search-frequency">
<option value="monthly">Monthly</option>
<option value="weekly">Weekly</option>
<option value="daily">Daily</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-monthly-additional">
<label for="saved-search-monthly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-monthly-on-day" class="no-border-panel-selector">
<option value="Sunday">The first Sunday</option>
<option value="Monday">The first Monday</option>
<option value="Tuesday">The first Tuesday</option>
<option value="Wednesday">The first Wednesday</option>
<option value="Thursday">The first Thursday</option>
<option value="Friday">The first Friday</option>
<option value="Saturday">The first Saturday</option>
<option value="day">The first day</option>
<option value="weekday">The first weekday</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-weekly-additional">
<label for="saved-search-weekly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-weekly-on-day" class="no-border-panel-selector saved-search-weekly-on-day">
<option value="Sunday">Sunday</option>
<option value="Monday">Monday</option>
<option value="Tuesday">Tuesday</option>
<option value="Wednesday">Wednesday</option>
<option value="Thursday">Thursday</option>
<option value="Friday">Friday</option>
<option value="Saturday">Saturday</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-report" class="action-panel-label">
Report format:
</label>
<select id="saved-search-report" class="no-border-panel-selector saved-search-report">
<option value="DocSum">Summary</option>
<option value="DocSumText">Summary (text)</option>
<option value="Abstract">Abstract</option>
<option value="AbstractText">Abstract (text)</option>
<option value="MEDLINE">PubMed</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-amount" class="action-panel-label">
Send at most:
</label>
<select id="saved-search-amount" class="no-border-panel-selector saved-search-amount">
<option value="1">1 item</option>
<option value="5" selected>5 items</option>
<option value="10">10 items</option>
<option value="20">20 items</option>
<option value="50">50 items</option>
<option value="100">100 items</option>
<option value="200">200 items</option>
</select>
</div>
<div>
<input type="checkbox" id="saved-search-send-if-no-result" class="saved-search-send-if-no-result" name="saved-search-send-if-no-result">
<label for="saved-search-send-if-no-result" class="action-panel-label smaller-checkbox">
Send even when there aren't any new results
</label>
</div>
<div class="action-panel-control-wrap option-text-in-email-wrap">
<label for="saved-search-email-text" class="action-panel-label">
Optional text in email:
</label>
<textarea name="saved-search-email-text"
id="saved-search-email-text"
class="saved-search-email-text"></textarea>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Saving..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Save
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your saved search' panel"
ref="linksrc=close_saved_search_panel"
aria-controls="saved-search-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="citation-manager-action-panel" class="citation-manager-action-panel action-panel" aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Create a file for external citation management software
</h2>
<form id="citation-manager-action-panel-form"
class="action-panel-content action-form"
action="/results-export-ids/"
data-by-search-action="/results-export-search-data/"
data-by-ids-action="/results-export-ids/"
method="post"
data-by-search-method="post"
data-by-ids-method="post">
<input type="hidden" name="csrfmiddlewaretoken" value="MBpyoDO79q6YbqK9nit6f2fg9drTisTL3o9D1thtJ1Ts5RQZ0B68JaLUHh95b3r5">
<input name="results-format" type="hidden" value="pubmed"/>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="save">
Create file
</button>
<button class="action-panel-cancel"
aria-label="Close 'Send citations to citation manager' panel"
ref="linksrc=close_citation_manager_panel"
aria-controls="citation-manager-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="rss-action-panel" class="rss-action-panel action-panel " aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your RSS Feed
</h2>
<form id="rss-action-panel-form"
class="rss-action-panel-form action-panel-content action-form"
data-create-rss-feed-url="/create-rss-feed-url/"
data-search-form-term-value="">
<input type="hidden" name="csrfmiddlewaretoken" value="MBpyoDO79q6YbqK9nit6f2fg9drTisTL3o9D1thtJ1Ts5RQZ0B68JaLUHh95b3r5">
<div class="action-panel-control-wrap">
<label for="rss-name" class="action-panel-label required-field-asterisk">
Name of RSS Feed:
</label>
<input maxlength="200"
placeholder="Name"
type="text"
name="rss-name"
id="rss-name"
class="rss-name"
value=''
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="rss-limit-wrap">
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="rss-limit" class="action-panel-label">
Number of items displayed:
</label>
<select id="rss-limit" class="no-border-panel-selector rss-limit">
<option value="5">5</option>
<option value="10">10</option>
<option value="15" selected="selected">15</option>
<option value="20">20</option>
<option value="50">50</option>
<option value="100">100</option>
</select>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Creating..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Create RSS
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your RSS' panel"
ref="linksrc=close_rss_panel"
aria-controls="rss-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
<div class="action-panel-control-wrap rss-link-copy-wrap">
<label for="rss-link" class="usa-sr-only">RSS Link</label>
<input placeholder="Your RSS Feed Link" type="text" name="rss-link" id="rss-link" class="rss-link" title="RSS Link">
<button
type="button"
disabled
class="rss-link-copy-button disabled"
data-ga-category="save_share"
data-ga-action="rss"
data-ga-label="copy">
Copy
</button>
</div>
</form>
</div>
</div>
</div>
</div>
<div class="article-page" id="article-page" data-article-pmid="15920477">
<aside class="page-sidebar">
<div class="inner-wrap">
<div class="full-text-links">
<div class="full-view">
<h3 class="title">
Full text links
</h3>
<div class="full-text-links-list">
<a class="link-item
dialog-focus"
href="https://doi.org/10.1038/sj.emboj.7600694"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=False&amp;PrId=3094&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=15920477&amp;db=pubmed&amp;nlmid=8208664"
title="See full text options at Nature Publishing Group"
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data-ga-action="Nature Publishing Group"
data-ga-label="15920477"
><img src="https://cdn.ncbi.nlm.nih.gov/corehtml/query/egifs/https:--www.embopress.org-pb-assets-embo-site-images-pubmed-embojnl_free.gif" alt="Nature Publishing Group full text link"><span class="text">
Nature Publishing Group
</span></a><a class="link-item
pmc
"
href="https://pmc.ncbi.nlm.nih.gov/articles/pmid/15920477/"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=True&amp;PrId=3494&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=15920477&amp;db=pubmed&amp;nlmid=8208664"
title="Free full text at PubMed Central"
data-ga-category="full_text"
data-ga-action="PMC"
data-ga-label="15920477"
><span class="text">
Free PMC article
</span></a>
</div>
</div>
<div class="short-view">
<a href="#" class="full-text-links-button full-text-links-dialog-trigger">
Full text links
</a>
</div>
</div>
<div class="actions-buttons sidebar"><h3 class="title">Actions</h3><div class="inner-wrap"><button class="citation-button citation-dialog-trigger"
aria-label="Open dialog with citation text in different styles"
data-ga-category="save_share"
data-ga-action="cite"
data-ga-label="open"
data-all-citations-url="/15920477/citations/"
data-citation-style="nlm"
data-pubmed-format-link="/15920477/export/"><span class="button-label">Cite</span></button><link type="text/css" href="ncbi-overlay-block/src/overlay-block.css"><div class="collections-button-container" data-article-id="15920477" data-article-db="pubmed"><button class="collections-button collections-dialog-trigger"
aria-label="Save article in MyNCBI collections."
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_button"
data-collections-open-dialog-enabled="false"
data-collections-open-dialog-url="https://account.ncbi.nlm.nih.gov/?back_url=https%3A%2F%2Fpubmed.ncbi.nlm.nih.gov%2F15920477%2F%23open-collections-dialog"
data-in-collections="false"><span class="button-label">Collections</span></button><div class="overlay" role="dialog"><div id="collections-action-dialog"
class="dialog collections-dialog"
aria-hidden="true"><div class="title">Add to Collections</div><div class="collections-action-panel action-panel"><form id="collections-action-dialog-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-myncbi-max-collection-name-length="100"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/"><input type="hidden" name="csrfmiddlewaretoken" value="MBpyoDO79q6YbqK9nit6f2fg9drTisTL3o9D1thtJ1Ts5RQZ0B68JaLUHh95b3r5"><div class="choice-group" role="radiogroup"><ul class="radio-group-items"><li><input type="radio"
id="collections-action-dialog-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_new"><label for="collections-action-dialog-new">Create a new collection</label></li><li><input type="radio"
id="collections-action-dialog-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_existing"><label for="collections-action-dialog-existing">Add to an existing collection</label></li></ul></div><div class="controls-wrapper"><div class="action-panel-control-wrap new-collections-controls"><label for="collections-action-dialog-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label><input
type="text"
name="add-to-new-collection"
id="collections-action-dialog-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="collections_button"
data-ga-action="create_collection"
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Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of the tyrosyl-3' phosphate linkage found in topoisomerase I-DNA covalent complexes. The inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1), is caused by a H493R mutation in Tdp1. Contrary to earlier proposals that this disease results from a loss-of-function mutation, we show here that this mutation reduces enzyme activity approximately 25-fold and importantly causes the accumulation of the Tdp1-DNA covalent reaction intermediate. Thus, the attempted repair of topoisomerase I-DNA complexes by Tdp1 unexpectedly generates a new protein-DNA complex with an apparent half-life of approximately 13 min that, in addition to the unrepaired topoisomerase I-DNA complex, may interfere with transcription and replication in human cells and contribute to the SCAN1 phenotype. The analysis of Tdp1 mutant cell lines derived from SCAN1 patients reveals that they are hypersensitive to the topoisomerase I-specific anticancer drug camptothecin (CPT), implicating Tdp1 in the repair of CPT-induced topoisomerase I damage in human cells. This finding suggests that inhibitors of Tdp1 could act synergistically with CPT in anticancer therapy.
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<p> Figure 1 </p>
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<div class="figure-caption-contents"><p> Tdp1 activity and protein in… </p></div>
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<p> Figure 1 </p>
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<div class="figure-caption-contents"><p> Tdp1 activity and protein in SCAN1 cells. ( <b> A </b> ) Sequencing gel analysis… </p></div>
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Figure 1
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<div class="figure-caption-contents">Tdp1 activity and protein in SCAN1 cells. (<b>A</b>) Sequencing gel analysis of Tdp1 activity assays with cell extracts from SCAN1 cells and related cell lines. Serial dilutions (10-fold) of extracts derived from cells that are either wt (wt-2, wt-5), homozygous for the H493R mutation (mut-1, mut-3, mut-4), or heterozygous at this locus (het-6, het-7) were incubated with substrate 12-Y, a 12-mer DNA oligonucleotide with a 3 tyrosine moiety. The product of the reaction is 12-P, a 12-mer with a 3 phosphate. Substrate 12-Y alone was loaded in lane 1. (<b>B</b>) Tdp1 assays of five-fold serial dilutions of recombinant wt and H493R Tdp1 proteins using substrate 12-Y. Substrate alone is shown in lane 1. The gel shown is representative of two independent experiments. (<b>C</b>) Immunoblot analysis of whole-cell extracts of the seven cell lines shown in (A). Rabbit anti-human Tdp1 antiserum was used to detect Tdp1. Purified recombinant His-tagged wt Tdp1 was loaded as a control in the far-right lane. (<b>D</b>) Specific inhibition of Tdp1 activity by anti-Tdp1 antiserum. Substrate 20-Topep, a 20-mer single-stranded DNA oligonucleotide with a small topoisomerase I-derived peptide attached to the 3 end, was used as the substrate (lane 1). The 20-P product of the Tdp1 reaction is shown in lane 2. In lanes 314, 10 μl of a 1:2 dilution of the indicated cell extract was tested for Tdp1 activity. Where indicated, the cell extracts were preincubated with anti-Tdp1 antiserum (lanes 710) or preimmune serum (lanes 1114) prior to the addition of substrate. A typical experiment of more than three independent experiments is shown.</div>
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<p> Figure 2 </p>
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<div class="figure-caption-contents"><p> H493R mutant accumulates the covalent… </p></div>
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<p> Figure 2 </p>
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<div class="figure-caption-contents"><p> H493R mutant accumulates the covalent Tdp1DNA reaction intermediate. ( <b> A </b> ) The indicated… </p></div>
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Figure 2
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<div class="figure-caption-contents">H493R mutant accumulates the covalent Tdp1DNA reaction intermediate. (<b>A</b>) The indicated Tdp1 enzyme was incubated with substrate 20-Topep. The conditions for the wt enzyme shown in lanes 1 and 2 were the same as shown in Figure 3, lane 6, the conditions for the H493R mutant shown in lanes 35 were the same as shown in Figure 3, lane 10, and the conditions for the H493A mutant shown in lanes 6 and 7 were the same as shown in Figure 3, lane 14. Reactions were stopped with SDS and a portion of each was digested with trypsin as indicated. The product resulting from trypsin treatment of the Tdp1DNA covalent intermediate is 20-Tdpep, and is predicted to consist of the 20-mer DNA with an 11-mer trypsin-resistant Tdp1 fragment attached to its 3 end. The sample in lane 5 was treated with proteinase K after trypsin treatment, which results in a 20-mer with a smaller peptide (labeled with a star). (<b>B</b>) A subset of the reactions shown in (A) was analyzed by SDSPAGE. Tdp1 proteins that are trapped in the reaction intermediate (20-Tdp) are radioactively labeled via the 5 end-labeled 20-mer DNA. Lanes 1 and 2, lanes 3 and 4, and lanes 5 and 6 correspond to lanes 1 and 2, lanes 3 and 4, and lanes 6 and 7 of (A), respectively. The mobility of two molecular weight markers is indicated on the right side of the gel. All experiments shown were repeated several times with identical results.</div>
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<p> Figure 3 </p>
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<div class="figure-caption-contents"><p> Activity assays of wt and… </p></div>
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<p> Figure 3 </p>
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<div class="figure-caption-contents"><p> Activity assays of wt and mutant forms of Tdp1. Five-fold serial dilutions of… </p></div>
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Figure 3
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<div class="figure-caption-contents">Activity assays of wt and mutant forms of Tdp1. Five-fold serial dilutions of the indicated proteins (starting at 1.4 μM) were incubated with substrate 20-Topep for 10 min, treated with trypsin, and analyzed on a sequencing gel. 20-Tdpep denotes the covalent reaction intermediate after trypsin treatment and 20-P the product of the cleavage reaction. One representative experiment of three is shown here.</div>
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<p> Figure 4 </p>
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<div class="figure-caption-contents"><p> Half-life of the covalent Tdp1… </p></div>
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<p> Figure 4 </p>
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<div class="figure-caption-contents"><p> Half-life of the covalent Tdp1 H493RDNA intermediate. ( <b> A </b> ) Tdp1 H493R and… </p></div>
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Figure 4
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<div class="figure-caption-contents">Half-life of the covalent Tdp1 H493RDNA intermediate. (<b>A</b>) Tdp1 H493R and 20-Topep were incubated for 2 min (zero time point, lane 2) before samples were taken at the indicated time points (lanes 210). Samples were treated with trypsin and analyzed in a sequencing gel. Lane 1 contains the substrate 20-Topep alone. (<b>B</b>) The decay of the Tdp1DNA covalent intermediate with time is plotted for the time-course samples shown in (A). Averages of three independent experiments are shown, with error bars representing standard deviations. Some error bars are too small to be seen.</div>
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<p> Figure 5 </p>
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<div class="figure-caption-contents"><p> CPT sensitivity of SCAN1 cell… </p></div>
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<p> Figure 5 </p>
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<div class="figure-caption-contents"><p> CPT sensitivity of SCAN1 cell lines. ( <b> A </b> ) Two wt cell lines… </p></div>
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Figure 5
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<div class="figure-caption-contents">CPT sensitivity of SCAN1 cell lines. (<b>A</b>) Two wt cell lines (wt-2, ▪; wt-5, ▴), two homozygous mutant SCAN1 cell lines (mut-3, □; mut-4, ▵), and one heterozygous cell line (het-6, ○) were grown in the presence of the indicated amounts of CPT and live cells were counted at 72 h. The percentage of live cells compared to the untreated control was determined as a function of the CPT concentration. This particular analysis was performed in triplicate and the results are shown with error bars representing the standard deviation. (<b>B</b>) The percentage of live cells is plotted versus time at 2.5 nM CPT for the experiment shown in (A).</div>
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<p> Figure 6 </p>
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<div class="figure-caption-contents"><p> Multiparameter flow cytometry analysis of… </p></div>
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<p> Figure 6 </p>
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<div class="figure-caption-contents"><p> Multiparameter flow cytometry analysis of cell survival, cell death, and early apoptosis. (AC)… </p></div>
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Figure 6
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<div class="figure-caption-contents">Multiparameter flow cytometry analysis of cell survival, cell death, and early apoptosis. (AC) Two wt cell lines (wt-2, ▪; wt-5, ▴) and two SCAN1 cell lines (mut-3, □; mut-4, ▵) were grown in the presence of the indicated amounts of CPT. The relative numbers of live, dead, and early apoptotic cells were determined at 48 h. Each data point shows the mean and standard deviation of triplicate samples. (<b>A</b>) The percentage of live treated cells as compared to live untreated cells at each data point is plotted as a function of the CPT concentration. (<b>B</b>) The difference between the percentage of dead cells in the treated cell population and the percentage of dead cells in the untreated control population is plotted as a function of CPT concentration. (<b>C</b>) The difference between the percentage of early apoptotic cells in the treated cell population and the percentage of apoptotic cells in the untreated control population is plotted as a function of CPT concentration.</div>
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<div class="figure-caption-contents"><p> Cell cycle distribution of live… </p></div>
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<div class="figure-caption-contents"><p> Cell cycle distribution of live and early apoptotic wt and SCAN1 mutant cell… </p></div>
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Figure 7
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<div class="figure-caption-contents">Cell cycle distribution of live and early apoptotic wt and SCAN1 mutant cell lines. (<b>A</b>) Cells were either untreated or treated with 5 nM CPT for 24 h. The abscissas represent the DNA content per cell as measured by the fluorescence intensity of Hoechst 33342, and reflect the G1, S, and G2 states of the cell cycle as indicated. The ordinates represent cell numbers. (<b>B</b>) Analysis is the same as described for the 5 nM CPT treatment in (A), except that cell numbers indicate those cells with low SYTO11 staining reflective of early apoptosis. (<b>C</b>) Percentage of cells in S phase after a 24-h treatment with the indicated concentrations of CPT (average of three experiments).</div>
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<div id="related-links" class="related-links">
<h2 class="title">
Related information
</h2>
<ul class="related-links-list"><li><a title="PubMed links associated with Books" href="https://www.ncbi.nlm.nih.gov/books?linkname=pubmed_books_refs&amp;from_uid=15920477"
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Cited in Books
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ClinVar
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Gene
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Gene (GeneRIF)
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MedGen
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MedGen (Bookshelf cited)
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Protein
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Protein (RefSeq)
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