我们感谢所有当前和以前的安德鲁·库恩克尔实验室成员的工作和讨论与协议和重复使用的数据在这里提出。这项工作得到了 ES065070 的项目 Z01 的支持, T.A.K. 国家卫生研究院 (NIH) 的内部研究司 (NIEHS)。

1. 碱性水解和链特定的南方印迹 (图 1)
<ol><li>细胞生长与基因组 DNA 分离<ol>
<li>根据制造商的说明, 使用酵母 DNA 纯化试剂盒分离酵母基因组 dna。使用处于生长指数阶段的区域性 (介于0.5 和1之间的光密度之间)。并用重悬在35毫升的 1x TE 缓冲器 (10 毫米三盐酸, pH 7.5, 1 毫米乙二胺乙酸 (EDTA)) 的最终 DNA 颗粒。</li>
		<li>添加1毫升的5毫克/毫升 RNase A 到 DNA 和孵育30分钟在37摄氏度。</li>
		<li>用0.1 容量的3米醋酸钠 (pH 5.2) 和2.5 卷100% 乙醇在冰上沉淀 DNA。</li>
		<li>离心机在 1.6万 x g 20 分钟在4摄氏度。用吸管取出上清液。</li>
		<li>用500毫升70% 乙醇清洗颗粒。用吸管取出上清液。</li>
		<li>用35毫升的 1x TE 缓冲器将并用重悬上的颗粒干燥。</li>
		<li>定量使用 fluorimeter (材料表) 与 dsDNA BR 试剂盒进行 DNA 检测。</li>
		<li>5毫克的 DNA 从每个样品使用0.1 容量3米醋酸钠, pH 5.2 和2.5 容量100% 乙醇为总容量200毫升 (增加 H2O 如果必要, 需要另外的5毫克脱氧核糖核酸, 如果中性凝胶控制是必要的)。在冰上孵育20分钟。</li>
		<li>离心机在 1.6万 x g 20 分钟在4摄氏度。用吸管取出上清液。</li>
		<li>把球团干在板凳上。并用重悬的 DNA 在14毫升的 H2O。</li>
	</ol></li>
	<li>碱性水解与凝胶电泳<ol>
<li>添加6毫升1米的 KOH 和孵育55°c 2 小时。</li>
		<li>在冰上孵化样品5分钟。</li>
		<li>添加4毫升6x 碱性 DNA 加载缓冲器: 300 毫米 KOH, 6 毫米 EDTA, pH 8.0, 18% 聚蔗糖 (材料表), 0.15% 甲酚绿色, 0.25% 二甲苯蓝 FF。</li>
		<li>铸造碱性凝胶, 由1% 琼脂糖, 50 毫米氢氧化钠, 1 毫米 EDTA, pH 8.0。使用一个梳子, 允许24毫升的 DNA 样本, 每车道加载。</li>
		<li>在室温下运行凝胶1x 碱性电泳缓冲器:50 毫米氢氧化钠, 1 毫米 EDTA, pH 8.0。包括适当的 DNA 大小标记。</li>
		<li>简单地向下旋转管子, 将整个24毫升的样品装入碱性琼脂糖凝胶中。Electrophorese 为30分钟在 30 V, 其次是 18-20 h 在 10 v。</li>
		<li>2孵化的凝胶在室温下与温和的搅拌在中和缓冲 I: 1 米三盐酸, 1.5 米氯化钠。</li>
		<li>将中性凝胶浸泡在200毫升水中, 其中含有 1/1万稀释的 SYBR 金染色, 室温下以温和摇动的方法对 DNA 进行染色。</li>
		<li>使用紫外线 transilluminator 可视化 DNA。增加碱敏感性导致小 dna 片段的出现, 表明在 dna5中失修 ribonucleotides 的密度较高。</li>
	</ol></li>
	<li>南部转移<ol>
<li>用毛细管动作10将碱性凝胶中的 DNA 转移到正电荷的尼龙膜上。理查德·山姆布鲁克和罗素的手册10中提供了如何设置此转移的示例。</li>
		<li>碱性转移缓冲器室温下浸泡碱性凝胶15分钟: 0.4 米氢氧化钠, 1 米氯化钠。</li>
		<li>湿尼龙膜 (切到胶粒的大小) 与水简单, 然后浸泡5分钟碱性转移缓冲器。</li>
		<li>通过在玻璃烘烤盘中反转3玻璃烧杯 (100 毫升), 建立转移平台。这道菜应该比琼脂糖凝胶的尺寸稍大一些。把玻璃盘子放在上面。</li>
		<li>在玻璃板上悬垂2大块3毫米的色谱纸 (切到凝胶的宽度大小和可以在边缘上悬垂的更长的边)。</li>
		<li>将碱性转移缓冲液倒入色谱纸上, 半填充玻璃碟。用5毫升的玻璃吸管平滑气泡。</li>
		<li>将琼脂糖凝胶放在顶部, 再平滑气泡。用 parafilm 包围凝胶的所有边缘。将少量碱性转移缓冲液倒入凝胶上。</li>
		<li>将预浸泡过的尼龙膜放在凝胶的顶部。把气泡弄平。</li>
		<li>湿2张纸切到大小的琼脂糖凝胶。将它们放在膜的顶端, 并平滑出任何气泡。</li>
		<li>在转移三明治的顶端放置一大堆纸巾 (切到比琼脂糖凝胶稍大一点的尺寸)。小心地将玻璃盘子放在纸巾上。将加权对象添加到顶部 (一本质量为400克的书工作良好)。</li>
		<li>让转移在室温下通宵进行。</li>
		<li>小心地把转移栈分开, 在室温下将膜浸泡15分钟, 中和缓冲 II: 0.5 米三盐酸盐, pH 7.2, 1 米氯化钠。</li>
		<li>用紫外交联剂将 DNA 与带电的尼龙膜交交。将膜放在交叉链接器中, 并使用 autocrosslink 设置。</li>
	</ol></li>
	<li>南方探头准备 注意: 此处描述的用于检测 DNA 链中初生的前导与滞后链中碱敏感点的方法基于以前发布的协议11。在我们的实验中 , 探测是在URA3报告基因上进行的 , 它入到与ARS306复制起源 (图 3A) 相邻的两个方向 ( OR1 或 OR2 ) 之一。在酵母基因组 DNA 中存在碱敏感点是核苷酸的一个指标, 允许使用 ribonucleotides 作为 DNA 聚合酶活性12、13、14的生物标志物。这种方法, 使用的目标 DNA 序列相邻的有效ARS306起源的复制, 应服从其他基因组的位置和目标基因, 以及。<ol>
<li>PCR-放大URA3报告基因的520基对段使用以下引物对: URA3-F1 (5´ GCTACATATAAGGAACGTGCTGC) 和 URA3-R1 (5´ CTTTGTCGCTCTTCGCAATGTC)。</li>
		<li>用 10-20 ng 酵母基因组 DNA 作为模板进行 PCR 反应, 4 ul 每底漆 (10 微米股票), 10 ul 10x 前 Taq 缓冲器 (Mg2 +加), 8 ul dNTP 混合物 (2.5 毫米每), 0.5 ul 的前 Taq DNA 聚合酶 (5 U/ul), 并调整最终音量为 100 ul 与 H2O。</li>
		<li>运行以下 PCR 程序:95 °c 为5分钟;30周期95°c 为1分钟, 55 °c 为1分钟, 72 °c 为2分钟;72°c 为10分钟并且保持在4°c。</li>
		<li>用 pcr 纯化试剂盒提纯 pcr 产物, 用50毫升的 H2O 洗脱 DNA。现在可以将其用作 radiolabeling 反应中的模板。</li>
		<li>使用以下引物为链特定的 radiolabeling: 使用 URA3-A (5´ CTCATCCTAGTCCTGTTGCTGCC), 以可视化的 DNA, 退火探测 A。这对应于新生的领先链 dna, 当URA3是在 OR2 和 tnascent 滞后链 dna 时URA3在 OR1。使用 URA3-B (5´-CAGTACCCTTAGTATATTCTCCAG) 可视化新生 DNA, 退火探针 B。这相当于新生的领先链 dna 时, 当URA3是在 OR1 和新生的滞后链 dna 时, URA3在 OR2 (图 3)。<ol>
<li>使用 10-20 ng 放大URA3片段作为模板 DNA 进行 radiolabeling 反应, 2 底漆 ul (10 微米库存), 5 ul 10x 前 Taq 缓冲器 (Mg2 +加), 4 毫米 dNTPs (减去 dCTP), 2.5 ul (5 μCi) 的一个 50 P dCTP, 0.5 ul ExTaq 脱氧核糖核酸聚合酶 (5 U/ul) 和调整最后容量到 50 ul 与 H2o 运行以下程序为 radiolabeling:94 °c 为5分钟;25周期94°c 为1分钟, 55 °c 为三十年代, 72 °c 为1分钟;72°c 为5分钟并且保持在4°c。</li>
		</ol>
</li>
		<li>使用 G-25 旋转列删除未合并的放射性标签。在添加核素样品之前, 将该列离心1分钟, 在 720 x g 和 removethe 缓冲器吹打。负载核素反应产品和离心机2分钟在 720 x g 洗脱标记探针。</li>
		<li>在95摄氏度孵育5分钟, 在杂交前立即变性探针。在冰上短暂冷却。</li>
	</ol></li>
	<li>南方杂交<ol>
<li>将润湿膜滚入杂交管中, 加入25毫升新制备的杂交缓冲液: 0.5 米磷酸钠缓冲液、pH 7.2、7% 月桂酸钠 (SDS)、1% 牛血清白蛋白 (BSA)。孵育在65°c 1-2 h 与旋转在一个杂交烤箱。</li>
		<li>小心地倒出溶液, 加入25毫升的杂交缓冲器加上 radiolabelled 探针。65摄氏度孵育 16-18 小时, 旋转。</li>
		<li>将溶液倒入放射性废物容器中。在室温下用磷酸盐-SDS 洗涤液进行5洗涤5分钟:40 毫米磷酸钠缓冲液, pH 7.2, 5% SDS, 0.5% BSA, 1 毫米 EDTA, pH 8.0。</li>
		<li>执行2洗涤15分钟每65°c 与磷酸盐 SDS 洗涤解决方案 II:40 毫米磷酸钠缓冲液, ph 7.2, 1% SDS, 1 毫米 EDTA, ph 值8.0。</li>
		<li>使用镊子将膜从杂交管中取出, 并在打开的塑料套筒保护器中放置。覆盖并暴露在成像板上。尝试多种不同的曝光时间, 从4小时到4维不等。</li>
		<li>如有必要, 用不同探针对印迹进行剥离和重新探测, 大约 3-4 次。在25毫升的50% 甲酰胺, 2x SSPE 溶液中, 将膜培养为2小时65摄氏度。 注: 20x SSPE 库存解决方案: 3 米氯化钠, 0.2 米 NaH2PO4, 0.02 米 EDTA, pH 7.4。</li>
		<li>将溶液倒入放射性废物容器中。在磷酸盐-SDS 洗涤液中, 每65°c 执行2次洗涤15分钟。</li>
		<li>检查膜与盖格计数器, 并重复剥离协议, 如果信号仍然存在。</li>
		<li>执行前述杂交和杂交。</li>
	</ol></li>
</ol>2.酵母菌株的变异率和特异性测定
<ol><li>准备细胞培养<ol>
<li>接种一个单一的殖民地 (健康酵母细胞需要 2-3 天在30°c 形成适当大小的殖民地) 生长在 YPD 琼脂补充与100毫克/毫升腺嘌呤入5毫升 YPDA 汤补充与250毫克/毫升腺嘌呤 (补充腺嘌呤是仅必要的, 如果使用的菌株是腺嘌呤营养缺陷型)。生长在一个振动筛孵化器或在30°c 的转子, 直到文化达到饱和 (36-48 小时的菌株, 没有严重的生长缺陷)。</li>
		<li>将单元格向下旋转 2000 x g, 5 分钟。</li>
		<li>丢弃上清液, 用5毫升的无菌水冲洗细胞。</li>
		<li>并用重悬500毫升的无菌水中的细胞。</li>
	</ol>
</li>
	<li>细胞培养和电镀的稀释<ol>
<li>对于非选择控制, 将96井板中的细胞以160万的因子和每种培养基100毫升的系数稀释到完整的 (COM) 板上。</li>
		<li>为了选择ura3突变体, 将100毫升未稀释的细胞放在 5 FOA 板上, 用于野生型 (应变)。如果预期的菌株的突变率高于某一应变, 则应相应稀释细胞。</li>
		<li>为can1突变体选择, 稀释细胞5倍和板100毫升到一个 canavanine 的板 (可以) 的一个重量应变。稀释系数为0.5。</li>
		<li>孵育板材在30°c 直到酵母殖民地形成。对于没有生长缺陷的菌株, 2-3 天通常是足够的 COM 和罐板和 3-5 天为 5 FOA 板。为了比较不同菌株之间的突变率, 最好选择同一天的生长来计算菌落数。把盘子放在冷房间里 (摄氏4摄氏度)。</li>
	</ol>
</li>
	<li>自发突变率的计算<ol>
<li>计算每个板块上可见的菌落数并记下菌落数</li>
		<li>用德雷克公式计算突变率: ÷ ( μNt) 。f是根据最终培养的细胞数除以突变体数量计算出的突变频率。Nt 是最终文化中的细胞数, μ是突变率。该方程可以用牛顿-拉夫逊法等迭代法求解。还有其他估计方法, 包括李和库尔森测试, 以计算突变率15。</li>
		<li>计算假设对数正态分布的 utation 速率的置信区间。最常用的是95% 置信区间。</li>
	</ol>
</li>
	<li>变异谱分析<ol>
<li>选择一个单一的殖民地从每个 5 FOA 或可以板块和刷新 YPDA 板块。</li>
		<li>执行菌落 pcr 放大URA3或CAN1基因和序列, 以检测突变使用以下引物: URA3-F (5 '-CGCATATGTGGTGTTGAAGAA-3 ') 和 URA3-R (5 '-TGTGAGTTTAGTATACATGCA-3 ') 的 pcr 扩增和800 bp URA3基因的测序;CAN1-F (5 '-CTTCTACTCCGTCTGCTTTC-3 ') 和 CAN1-R (5 '-CAGAGTTCTTCAGACTTC-3 ') 为放大和 CAN1-AR (5 '-TGAAATGTGAAGGCAGCGTT-3 '), CAN1-9R (5 '-CCTGCAACACCAGTGATA-3 '), CAN1-10R (5 '-GAGGATGTAACAGGGATGAAT-3 ') 和 CAN1-BR (5 '-CGGTGTATGACTTATGAGGG-3 ') 用于测序的 1800 bp CAN1报告基因。</li>
		<li>为了进行菌落 PCR, 并用重悬在 5 ul2O 的一个单一的殖民地, 并使用 1 ul 作为模板。添加 1 ul 每一个底漆 (5 微米股票), 2 ul 5x KAPA2G 缓冲器 (Mg2 +加), 0.5 ul dNTP (2.5 毫米), 0.5 ul KAPA2G 快速 DNA 聚合酶, 12 ul 的 H2O。</li>
		<li>对于URA3基因的扩增, 运行以下 PCR 程序:95 °c 5 分钟;5周期95°c 为十年代, 61 °c 十五年代, 72 °c 为三十年代;35周期95°c 为十年代, 61 °c 十五年代, 72 °c 为三十年代;72°c 为2分钟并且保持在4°c。对于CAN1基因的扩增, 运行以下 PCR 程序:95 °c 5 分钟;5周期95°c 为十年代, 61 °c 十五年代, 72 °c 为四十五年代;35周期95°c 为十年代, 61 °c 十五年代, 72 °c 为四十五年代;72°c 为2分钟并且保持在4°c。</li>
		<li>使用程序 (如 DNASTAR Seqman Pro 或 Sequencher) 将排序结果与参考序列对齐以识别突变。</li>
		<li>按突变类型 (图 4) 或突变位置 (图 5) 编译突变数据。根据突变的频率计算特定的突变率 (观察到的事件数除以突变序列的数量) 乘以总的突变率。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Joyce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+the+right+sugar:+how+polymerases+select+a+nucleotide+substrate.">Choosing the right sugar: how polymerases select a nucleotide substrate.</a> Proceedings of the National Acaddemy of Sciences U S A. 94 (5), 1619-1622 (1997).</li><li>Nick McElhinny, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundant+ribonucleotide+incorporation+into+DNA+by+yeast+replicative+polymerases.">Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases.</a> Proceedings of the National Acaddemy of Sciences U S A. 107 (11), 4949-4954 (2010).</li><li>Sparks, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNase+H2-initiated+ribonucleotide+excision+repair.">RNase H2-initiated ribonucleotide excision repair.</a> Molecular Cell. 47 (6), 980-986 (2012).</li><li>Li, Y., Breaker, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+RNA+Degradation+by+Specific+Base+Catalysis+of+Transesterification+Involving+the+2'-Hydroxyl+Group.">Kinetics of RNA Degradation by Specific Base Catalysis of Transesterification Involving the 2'-Hydroxyl Group.</a> Journal of the American Chemical Society. 121 (23), 5364-5372 (1999).</li><li>Nick McElhinny, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+instability+due+to+ribonucleotide+incorporation+into+DNA.">Genome instability due to ribonucleotide incorporation into DNA.</a> Nature Chemical Biology. 6 (10), 774-781 (2010).</li><li>Williams, J. S., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonucleotides+in+DNA:+origins,+repair+and+consequences.">Ribonucleotides in DNA: origins, repair and consequences.</a> DNA Repair (Amst). 19, 27-37 (2014).</li><li>Williams, J. S., Lujan, S. A., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+ribonucleotides+incorporated+during+eukaryotic+DNA+replication.">Processing ribonucleotides incorporated during eukaryotic DNA replication.</a> Nature Reviews Molecular Cell Biology. 17 (6), 350-363 (2016).</li><li>Jones, M. E., Thomas, S. M., Rogers, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luria-Delbruck+fluctuation+experiments:+design+and+analysis.">Luria-Delbruck fluctuation experiments: design and analysis.</a> Genetics. 136 (3), 1209-1216 (1994).</li><li>Zheng, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+practical+guide+to+the+Luria-Delbruck+protocol.">A new practical guide to the Luria-Delbruck protocol.</a> Mutat Research. 781, 7-13 (2015).</li><li>Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: a Laboratory Manual. , (2001).</li><li>Miyabe, I., Kunkel, T. A., Carr, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+roles+of+DNA+polymerases+epsilon+and+delta+at+the+eukaryotic+replication+fork+are+evolutionarily+conserved.">The major roles of DNA polymerases epsilon and delta at the eukaryotic replication fork are evolutionarily conserved.</a> PLoS Genetics. 7 (12), e1002407 (2011).</li><li>Lujan, S. A., Williams, J. S., Clausen, A. R., Clark, A. B., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonucleotides+are+signals+for+mismatch+repair+of+leading-strand+replication+errors.">Ribonucleotides are signals for mismatch repair of leading-strand replication errors.</a> Molecular Cell. 50 (3), 437-443 (2013).</li><li>Williams, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topoisomerase+1-mediated+removal+of+ribonucleotides+from+nascent+leading-strand+DNA.">Topoisomerase 1-mediated removal of ribonucleotides from nascent leading-strand DNA.</a> Molecular Cell. 49 (5), 1010-1015 (2013).</li><li>Clausen, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+replication+enzymology+in+vivo+by+genome-wide+mapping+of+ribonucleotide+incorporation.">Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation.</a> Nature Structural &amp; Molecular Biology. 22 (3), 185-191 (2015).</li><li>Foster, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+determining+spontaneous+mutation+rates.">Methods for determining spontaneous mutation rates.</a> Methods Enzymology. 409, 195-213 (2006).</li><li>Williams, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+processing+of+ribonucleotides+in+DNA+by+topoisomerase+1+is+leading-strand+specific.">Evidence that processing of ribonucleotides in DNA by topoisomerase 1 is leading-strand specific.</a> Nature Structural &amp; Molecular Biology. 22 (4), 291-297 (2015).</li><li>Pavlov, Y. I., Shcherbakova, P. V., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+consequences+of+putative+active+site+mutations+in+yeast+DNA+polymerases+alpha,+epsilon,+delta,+and+zeta.">In vivo consequences of putative active site mutations in yeast DNA polymerases alpha, epsilon, delta, and zeta.</a> Genetics. 159 (1), 47-64 (2001).</li><li>Nick McElhinny, S. A., Kissling, G. E., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+correction+of+lagging-strand+replication+errors+made+by+DNA+polymerases+{alpha}+and+{delta}.">Differential correction of lagging-strand replication errors made by DNA polymerases {alpha} and {delta}.</a> Proceedings of the National Acaddemy of Sciences U S A. 107 (49), 21070-21075 (2010).</li><li>Clark, A. B., Lujan, S. A., Kissling, G. E., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mismatch+repair-independent+tandem+repeat+sequence+instability+resulting+from+ribonucleotide+incorporation+by+DNA+polymerase+epsilon.">Mismatch repair-independent tandem repeat sequence instability resulting from ribonucleotide incorporation by DNA polymerase epsilon.</a> DNA Repair (Amst). 10 (5), 476-482 (2011).</li><li>Lujan, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mismatch+repair+balances+leading+and+lagging+strand+DNA+replication+fidelity.">Mismatch repair balances leading and lagging strand DNA replication fidelity.</a> PLoS Genetics. 8 (10), e1003016 (2012).</li><li>Reijns, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+removal+of+ribonucleotides+from+DNA+is+essential+for+mammalian+genome+integrity+and+development.">Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development.</a> Cell. 149 (5), 1008-1022 (2012).</li><li>Lujan, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+polymerase+fidelity+and+mismatch+repair+bias+genome+variation+and+composition.">Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition.</a> Genome Research. 24 (11), 1751-1764 (2014).</li></ol>

作者没有什么可透露的。

在这里, 我们描述了两组试验的协议, 这些实验经常用于对 DNA 复制过程中 ribonucleotides 的半定量分析, 以及失修 ribonucleotides 的诱变效应。虽然这些方法涉及模型真核细胞的酵母, 这些技术可以很容易地适应其他微生物, 甚至更高的真核生物。
碱性琼脂糖电泳失修 ribonucleotides 在 DNA 中的应用探讨与南方印迹相结合, 提供了关于 ribonucleotides 的数量和它们被纳入其中的链的重要信息。然而, 重要的是要注意, 碱敏感性也可能导致其他 DNA 病变, 如 abasic 的网站。另一种检测 DNA ribonucleotides 的方法是用纯化的 RNase H2 酶治疗, 就像哺乳动物细胞21中所做的那样。尽管我们的方法涉及在我们的突变分析实验中使用的URA3报告基因在新生的前导和滞后链中对碱敏感点进行探测, 但可以设计在其他位置进行退火的探头, 以便获取有关其他基因组站点核苷酸密度的重要信息。该过程的南方印迹部分也可作为其他常规 DNA 探测实验的参考。
利用波动试验测量突变率已被广泛应用于微生物中各种诱变事件的分析, 因此不限于分析 ribonucleotides 在 DNA 中的诱变效应。在这个实验中, 增加独立文化的数量可以大大提高测量的准确度。独立的殖民地可以通过裸奔一个兴趣的 YPDA 培养基或通过分析的独立孢子殖民地获得的四解剖的双倍酵母染色。理想情况下, 鼓励使用多个独立的单倍体酵母菌株 (例如, 从四解剖), 以尽量减少应变对应变变化的影响。当自发突变率较高或菌株有生长缺陷时, 这一点尤为重要。另外一种可以用来测量自发突变率的方法是突变积累实验22。在这一分析中, 突变频率是确定的细胞后, 广泛的增长, 可用于计算的突变率。突变积累实验与深测序技术的耦合已被证明是一个强有力的工具来分析基因组的突变率和特异性。

在真核 dna 复制过程中, ribonucleotides 由所有三主要 dna replicases、dna 聚合酶 (年 floss-pols) α、ε和δ1、2组成基因组。RNase H2-dependent 核苷酸切除修复 (3) 去除大多数这些嵌入式 ribonucleotides。
DNA 中的核苷酸易水解, 因为 2 ' 羟基的糖基团可以攻击相邻的磷酸二酯键, 释放一个末端与 2 '-3 ' 循环磷酸盐和另一个与 5 '-OH4。碱性条件可以大大加快这种反应。因此, 在碱性溶液的孵化过程中, 嵌入 ribonucleotides 的水解会导致基因组 DNA 的碎片化, 这可以通过碱-琼脂糖电泳5进行可视化。这种 DNA 可以转移到一个膜, 并通过南方印迹分析, 利用链特定的探针, 允许识别碱敏感的网站所造成的 ribonucleotides 纳入新生的领先或滞后链 DNA,分别。
在缺乏 RNase H2 活性的酵母细胞中, 当拓扑异构酶 I (Top1) 在嵌入的核苷酸6、7上刻痕 DNA 时, 可以启动 ribonucleotides 的去除。然而, 当 Top1 在 3 ' 侧核苷酸, 这产生 5 '-OH 和 2 '-3 ' 循环磷酸盐 DNA 末端是顽固的对 religation。不修复或异常处理这些 ' 肮脏的末端 ' 可能导致基因组不稳定。此外, 如果切口发生在重复 DNA 序列中, 修复过程可能导致删除突变。这对于串联重复是特别成问题的, 在 RNase H2-deficient 细胞中通常观察到短的删除 (介于两和五基对之间)。在 Top1-dependent 酵母 RNase H2 活性缺乏的情况下, DNA 聚合酶ε突变体 (pol2-M644G) 混杂在新生的主导链合成过程中会加剧核苷酸的有害影响。
DNA 中 ribonucleotides 的处理导致自发的突变, 这种突变可以通过适当的报告基因和选择相应的表型变化来检测。波动测试或 Luria 和德尔布吕克实验是最常用的方法之一, 以测量自发突变率使用可选择的报告基因8,9。在酵母中, URA3和CAN1基因可以作为一个正向突变检测的记者, 它允许检测所有导致基因功能丧失的突变类型。自发突变率被估计为多平行培养所观察到的中位数, 从单个菌落开始, 而目标报告基因中没有突变。酵母 RNase H2-deficient 菌株如rnh201Δ具有适度升高的整体自发突变率, 这主要是由于串联重复序列的 2-5 bp 删除率升高引起的。因此, 为了充分描述 ribonucleotides 在基因组中的诱变效应, 需要确定特定的突变率。在这种情况下, URA3或CAN1的报告基因可以被放大和排序, 以确定的类型和地点的突变, 和特定的突变率可以计算。在多个独立URA3或CAN1突变体中发现的突变可以用来产生突变谱。

<table><tbody><tr><td>YPD media</td><td></td><td></td><td>20 g dextrose, 20 g peptone, 10g yeast extract, in deionized H&lt;sub&gt;2&lt;/sub&gt;O up to 1 L, add 20 g Bacto agar for solid media, autoclave.</td></tr><tr><td>COM plates</td><td></td><td></td><td>1.3 g SC dropout mix, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar deionized H&lt;sub&gt;2&lt;/sub&gt;O up to 1 L. Adjust pH to 5.8. Autoclave and 30 &amp;ndash; 35 ml per plate.</td></tr><tr><td>CAN plates</td><td></td><td></td><td>1.3 g SC-Ura dropout powder, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar, 25 mg uracil and H2O up to 1 L. Autoclave for 15 mins at 121 &amp;deg;C and cool down to 56 &amp;deg;C. Add 6 mL of filter-sterilized 1% canavanine sulfate solution.</td></tr><tr><td>5-FOA plates</td><td></td><td></td><td>1.3 g SC-Ura dropout powder, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar, 25 mg uracil and H2O up to 800 mL. Autoclave for 15 mins at 121 &amp;deg;C and cool down to 60 &amp;deg;C. Add 200 mL of filter-sterilized 0.5% 5-FOA solution.</td></tr><tr><td>L-Canavanine sulfate</td><td>US Biological</td><td>C1050</td><td></td></tr><tr><td>5-FOA</td><td>US Biological</td><td>F5050</td><td></td></tr><tr><td>20 mL glass culture tube</td><td></td><td></td><td>Any brand</td></tr><tr><td>Culture rotator in 30 &amp;deg;C incubator</td><td></td><td></td><td>Shaker incubator can be used instead</td></tr><tr><td>96 well round bottom plates</td><td></td><td></td><td>Sterile, any brand</td></tr><tr><td>3 mm glass beads</td><td>Fisher Scientific</td><td>11-312A</td><td>Autoclave before use</td></tr><tr><td>12-channel pipettes</td><td></td><td></td><td>Any brand</td></tr><tr><td>Ex Taq DNA Polymerase</td><td>TaKaRa</td><td>RR001</td><td></td></tr><tr><td>Epicentre MasterPure Yeast DNA Purification Kit</td><td>Epicenter</td><td>MPY80200</td><td></td></tr><tr><td>3 M sodium acetate</td><td>Sigma-Aldrich</td><td>S7899</td><td></td></tr><tr><td>100% ethanol</td><td></td><td></td><td></td></tr><tr><td>Qubit 2.0 Fluorometer</td><td>Invitrogen</td><td>Q32866</td><td>Newer model available</td></tr><tr><td>dsDNA BR Assay kit</td><td>Invitrogen</td><td>Q32850</td><td></td></tr><tr><td>KOH</td><td>Sigma-Aldrich</td><td>221473</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E7889</td><td></td></tr><tr><td>Ficoll 400</td><td>Dot Scientific Inc.</td><td>DSF10400</td><td></td></tr><tr><td>Bromocresol green</td><td>Eastman</td><td>6356</td><td></td></tr><tr><td>Xylene cyanol FF</td><td>International Technologies Inc.</td><td>72120</td><td></td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>1 M Tris-HCl (pH 8.0)</td><td>Teknova</td><td>T5080</td><td></td></tr><tr><td>SYBR Gold Nucleic Acid Gel Stain</td><td>Invitrogen</td><td>S11494</td><td></td></tr><tr><td>UV transilluminator</td><td></td><td></td><td></td></tr><tr><td>Amersham Nylon membrane Hybond-N+</td><td>GE Healthcare</td><td>RPN303B</td><td></td></tr><tr><td>3 MM CHR Chromotography paper</td><td>Whatman</td><td>3030-392</td><td></td></tr><tr><td>NaCl</td><td>Caledon</td><td>7560-1</td><td></td></tr><tr><td>Stratalinker 1800</td><td>Stratagene</td><td></td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>G-25 spin column</td><td>GE Healthcare</td><td>27-5325-01</td><td></td></tr><tr><td>1 M Sodium phosphate buffer (pH 7.2)</td><td>Sigma-Aldrich</td><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (S9638);&lt;br/&gt; Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (S9390)</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L4522</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A3059</td><td></td></tr><tr><td>Formamide</td><td>Sigma-Aldrich</td><td>47671</td><td></td></tr><tr><td>Geiger counter</td><td></td><td></td><td></td></tr></tbody></table>

studying ribonucleotide incorporation: strand-specific detection of ribonucleotides in the yeast genome and measuring ribonucleotide-induced mutagenesis

ribonucleotides 在核 DNA 中的存在被证明是基因组不稳定的来源。核苷酸的范围可以用碱性水解和凝胶电泳来评定, 因为 RNA 在碱性条件下很容易水解。这与南方污点分析结合在一起可以用来确定 ribonucleotides 的位置和链。然而, 这一过程只是半定量的, 可能不够敏感, 以检测核苷酸内容的小变化, 虽然链特定的南方印迹探测提高灵敏度。作为衡量 ribonucleotides 在 DNA 中最显著的生物学后果之一, 自发诱变可以用正向突变法进行分析。使用适当的报告基因, 可以选择导致功能丧失的罕见突变, 并通过将波动实验数据与报告基因的 DNA 测序相结合来测量整体和特异的突变率。波动分析法适用于在特定的遗传背景或生长条件下检测各种诱变过程。

碱凝胶电泳法处理基因组 dna, 允许对 dna 碎片进行半定量检测, 因为 ribonucleotides 的丰度稳定。图 2显示了用或不带5KOH 治疗酵母基因组 DNA 的凝胶图像。Polε的催化亚单位 Pol2 的 M644L 变异降低了吸收 ribonucleotides 的能力, M644G 突变体的 ribonucleotides 比聚酶多。如《议定书》所述, 碱性凝胶可以通过链特定的南方印迹分析进一步探讨。随着对探测到的基因组站点相对于其相邻起源的了解, 我们可以有选择地探测 ribonucleotides 纳入初生的前导或滞后链。图 3显示了南部印迹结果探测URA3报告基因插入两个相反的方向接近早期射击复制起源, ARS30616。通过使用前导链特定探针, 我们观察了 ribonucleotides 中初生链 dna 中碱裂解引起的 dna 分裂模式, 并在表达聚合酶α、δ和ε的变种的应变核苷酸公司成立。
利用波动实验, 可以测量含有报告基因的酵母菌株的突变率。图 4显示了市建局3 和CAN1的突变率, 这些菌株表达了大量的聚合酶或pol2-M644G突变体, 有或没有功能 RNase H2。RNase H2 的缺乏导致了两种背景下整体突变率的适度增加。然而, 对突变特异性的仔细检查表明, 在缺乏 RNase H2 的菌株 (图 5) 中, 短的缺失的突变率有很强的增加, 特别是串联重复序列。图 5A比较了201Δ菌株和 rnh 的突变特异性,图 5B显示了进一步升高的核苷酸在初始的 DNA 链中的诱变效应。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58020/58020fig1.jpg"> 图 1: 核苷酸在酵母基因组 DNA 中加入的碱敏感部位的链特定检测所涉及的协议步骤.本程序涉及的各种主要步骤的概述, 从酵母基因组 DNA 分离开始, 并通过最后的南方印迹分析。<a href="https://www.jove.com/files/ftp_upload/58020/58020fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58020/58020fig2.jpg"> 图 2: 由碱性琼脂糖凝胶电泳获得的具有代表性的结果图像.这一数字已经从尼克 McElhinny,等等。5. 不同基因型菌株的酵母基因组 DNA 用氯化钾 (中性) 或 KOH (碱性) 治疗, 然后在中性或碱性琼脂糖凝胶上分离。"M644G" 和 "的" 是指的状态.<a href="https://www.jove.com/files/ftp_upload/58020/58020fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58020/58020fig3.jpg"> 图 3: 用南方印迹对碱性琼脂糖凝胶进行链式探测.这个数字已经从威廉斯,等等。13. ( A )核素探针退火到URA3报告基因中的新生的领先 DNA 链 , 已入接近ARS306在两个相反的方向 ( OR1 和 OR2 ) 。(B)南方印迹的代表性结果使用新生的领先的线特定探针。显示的是在一个应变与 DNA 聚合酶, 或pol1-L868M, pol2-M644G或pol3-L612M变种菌株与或没有 RNase H2 的新生领先链的探测结果。POL1、 POL2和POL3基因分别编码年 floss-pols α、ε和δ的催化亚基。变形是更加混杂的为核苷酸并网5,12,17。(C)从(B)每条车道的总数量中量化放射性信号。值以总计的百分比表示。<a href="https://www.jove.com/files/ftp_upload/58020/58020fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58020/58020fig4.jpg"> 图 4: 酵母菌株的自发突变率.这一数字已经从尼克 McElhinny,等等。5. URA3和CAN1报告基因用于正突变检测, 以测量所述菌株的突变率。URA3 记者在 "方向 2" (OR2,图 3A)。每个测量都包含95% 置信区间 (CI)。<a href="https://www.jove.com/files/ftp_upload/58020/58020fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58020/58020fig5.jpg"> 图 5: URA3-OR2 报告基因的突变谱.这一数字已从以前的出版物5、18、19、20中改编。在 804 bp URA3编码序列中, 描述了在正突变试验中选择的每个独立的 5-FOA 抗性群体中所观察到的位置和突变类型。字母表示基替换, 开放三角形表示单个基删除, 闭合三角形表示单个基插入, 而实线表示短删除。(A) rnh201Δ菌株的突变谱。在序列上面的红色标签是在小波中观察到的突变, 而下面的蓝色标签是在rnh201Δ菌株中观察到的。(B) pol2-M644G和pol2-M644G rnh201Δ菌株的突变谱。序列上方的红色标签是在pol2-M644G中观察到的突变, 而蓝色则低于pol2-M644G rnh201Δ菌株的序列。<a href="https://www.jove.com/files/ftp_upload/58020/58020fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis at Jo

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

Ribonucleotides 是在核 DNA 复制过程中纳入基因组的最丰富的非规范核苷酸之一。如果没有正确去除, ribonucleotides 会导致 DNA 损伤和突变。在这里, 我们提出了两个实验方法, 用于评估核苷酸的丰度纳入 DNA 及其诱变效应。

研究核苷酸合并: 酵母基因组 Ribonucleotides 的链特异检测及核苷酸诱变的检测

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...

studying-ribonucleotide-incorporation-strand-specific-detection

Nanyang Technological University

Research

JoVE Journal

Biology

7.7K 次观看.  National Institute of Environmental Health Sciences, NIH, DHHS. Ribonucleotides 是在核 DNA 复制过程中纳入基因组的最丰富的非规范核苷酸之一。如果没有正确去除, ribonucleotides 会导致 DNA 损伤和突变。在这里, 我们提出了两个实验方法, 用于评估核苷酸的丰度纳入 DNA 及其诱变效应。

视频: Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

核糖体剖面对芽殖酵母中翻译的全基因组定量

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

科研

生物化学

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

遗传学

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

酿酒酵母4-硫尿嘧啶代谢标记及新合成 mRNA 作为 RNA 聚合酶 II. 活性的一种代理

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

生物学

DNAzyme-dependent Analysis of rRNA 2&#8217;-O-Methylation

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2&#8217;-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2&#8217;-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-&#8217;O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2&#8217;-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2&#8217;-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.

Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2&#8217;-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

rRNA 2'-O-甲基化的脱氧核糖核酸依赖性分析

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in ...

发育生物学

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Mutation rates in young Saccharomyces cerevisiae cells measured through fluctuation tests are used to predict mutation frequencies for mother cells of different replicative ages. Magnetic sorting and flow cytometry are then used to measure actual mutation frequencies and age of mother cells to identify any deviations from predicted mutation frequencies.

结合母细胞和波动测试磁分选，以分析基因组不稳定性的过程中有丝分裂细胞衰老<em&gt;酿酒酵母</em

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA的衰变率的测量<em&gt;酿酒酵母</em&gt;使用<em&gt; rpb1-1</em&gt;株

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

化学遗传相互作用的快速鉴定<em&gt;酿酒酵母</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

人线粒体 DNA 中核苷酸的同时定位与定量

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae

Eukaryotic DNA replication is a highly regulated process that ensures that the genetic blueprint of a cell is correctly duplicated prior to chromosome segregation. As DNA synthesis defects underlie chromosome rearrangements, monitoring DNA replication has become essential to understand the basis of genome instability. Saccharomyces cerevisiae is a classical model to study cell cycle regulation, but key DNA replication parameters, such as the fraction of cells in the S phase or the S-phase duration, are still difficult to determine. This protocol uses short and non-toxic pulses of 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analog, in engineered TK-hENT1 yeast cells, followed by its detection by Click reaction to allow the visualization and quantification of DNA replication with high spatial and temporal resolution at both the single-cell and population levels by microscopy and flow cytometry. This method may identify previously overlooked defects in the S phase and cell cycle progression of yeast mutants, thereby allowing the characterization of new players essential for ensuring genome stability.

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae

We describe two complementary protocols to accurately determine S-phase duration in S. cerevisiae using EdU, a thymidine analog, which is incorporated in vivo and detected using Click chemistry by microscopy and flow cytometry. It allows for the easy characterization of the duration of DNA replication and overlooked replication defects in mutants.

使用酿酒酵母中的5-乙炔基-2'-脱氧尿苷掺入测定S相持续时间

Eukaryotic DNA replication is a highly regulated process that ensures that the genetic blueprint of a cell is correctly duplicated prior to chromosome ...

研究核苷酸合并: 酵母基因组 Ribonucleotides 的链特异检测及核苷酸诱变的检测

摘要

探索更多视频