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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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)。
Materials
| YPD media | 20 g dextrose, 20 g peptone, 10g yeast extract, in deionized H2O up to 1 L, add 20 g Bacto agar for solid media, autoclave. | ||
| COM plates | 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 H2O up to 1 L. Adjust pH to 5.8. Autoclave and 30 – 35 ml per plate. | ||
| CAN plates | 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 °C and cool down to 56 °C. Add 6 mL of filter-sterilized 1% canavanine sulfate solution. | ||
| 5-FOA plates | 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 °C and cool down to 60 °C. Add 200 mL of filter-sterilized 0.5% 5-FOA solution. | ||
| L-Canavanine sulfate | US Biological | C1050 | |
| 5-FOA | US Biological | F5050 | |
| 20 mL glass culture tube | Any brand | ||
| Culture rotator in 30 °C incubator | Shaker incubator can be used instead | ||
| 96 well round bottom plates | Sterile, any brand | ||
| 3 mm glass beads | Fisher Scientific | 11-312A | Autoclave before use |
| 12-channel pipettes | Any brand | ||
| Ex Taq DNA Polymerase | TaKaRa | RR001 | |
| Epicentre MasterPure Yeast DNA Purification Kit | Epicenter | MPY80200 | |
| 3 M sodium acetate | Sigma-Aldrich | S7899 | |
| 100% ethanol | |||
| Qubit 2.0 Fluorometer | Invitrogen | Q32866 | Newer model available |
| dsDNA BR Assay kit | Invitrogen | Q32850 | |
| KOH | Sigma-Aldrich | 221473 | |
| EDTA | Sigma-Aldrich | E7889 | |
| Ficoll 400 | Dot Scientific Inc. | DSF10400 | |
| Bromocresol green | Eastman | 6356 | |
| Xylene cyanol FF | International Technologies Inc. | 72120 | |
| NaOH | Sigma-Aldrich | S8045 | |
| 1 M Tris-HCl (pH 8.0) | Teknova | T5080 | |
| SYBR Gold Nucleic Acid Gel Stain | Invitrogen | S11494 | |
| UV transilluminator | |||
| Amersham Nylon membrane Hybond-N+ | GE Healthcare | RPN303B | |
| 3 MM CHR Chromotography paper | Whatman | 3030-392 | |
| NaCl | Caledon | 7560-1 | |
| Stratalinker 1800 | Stratagene | ||
| QIAquick PCR Purification Kit | Qiagen | 28106 | |
| G-25 spin column | GE Healthcare | 27-5325-01 | |
| 1 M Sodium phosphate buffer (pH 7.2) | Sigma-Aldrich | NaH2PO4 (S9638); Na2HPO4 (S9390) |
|
| SDS | Sigma-Aldrich | L4522 | |
| BSA | Sigma-Aldrich | A3059 | |
| Formamide | Sigma-Aldrich | 47671 | |
| Geiger counter |
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