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Modification of 3’ Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity
利用DNA聚合酶θ末端转移酶活性修饰DNA和RNA的3'末端   

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Abstract

DNA polymerase θ (Polθ) is a promiscuous enzyme that is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ). During this form of DSB repair, Polθ performs terminal transferase activity at the 3’ termini of resected DSBs via templated and non-templated nucleotide addition cycles. Since human Polθ is able to modify the 3’ terminal ends of both DNA and RNA with a wide array of large and diverse ribonucleotide and deoxyribonucleotide analogs, its terminal transferase activity is more useful for biotechnology applications than terminal deoxynucleotidyl transferase (TdT). Here, we present in detail simple methods by which purified human Polθ is utilized to modify the 3’ terminal ends of RNA and DNA for various applications in biotechnology and biomedical research.

Keywords: DNA polymerase(DNA聚合酶), DNA repair(DNA修复), DNA modification(DNA修饰), Alternative end-joining(替代末端连接), Terminal deoxynucleotidyl transferase(末端脱氧核苷酸转移酶), Biotechnology(生物技术), Nucleotide analogs(核苷酸类似物)

Background

The human POLQ gene encodes a large protein that contains an N-terminal superfamily 2 (SF2) type helicase domain and a C-terminal A-family polymerase domain (Sfeir and Symington, 2015; Black et al., 2016; Wood and Doublie, 2016). The protein also encodes for a large central domain whose function has yet to be ascribed. Polθ is expressed in metazoans and has been shown to function in multiple aspects of DNA replication and repair (Black et al., 2016; Wood and Doublie, 2016). Recent work showed that mammalian Polθ is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ), also known as microhomology mediated end-joining (MMEJ) (Yousefzadeh et al., 2014; Kent et al., 2015; Mateos-Gomez et al., 2015). This essential function of the polymerase is conserved among metazoans (Chan et al., 2010; Koole et al., 2014).

Interestingly, Polθ mediated alt-EJ results in relatively large deletions and insertions (indels) at DNA repair junctions compared to the more accurate non-homologous end-joining (NHEJ) pathway (Black et al., 2016). For instance, alt-EJ typically generates insertions ranging from 1-6 base pairs (bp), and in some cases insertions can exceed 30 bp (Yousefzadeh et al., 2014; Mateos-Gomez et al., 2015; Black et al., 2016; Kent et al., 2016). Intriguingly, multiple studies from invertebrates and vertebrate systems show that some insertion tracts are templated by nearby DNA sequences such as those flanking the DSB (Black et al., 2016). In other cases, insertion sequences appear to be random (Black et al., 2016). These and other studies led to the idea that Polθ might generate insertion tracts at DSBs by both templated and non-templated terminal transferase mechanisms.

Indeed, in a recent study Kent et al. demonstrated that the human Polθ polymerase domain, hereinafter referred to as Polθ, exhibits robust terminal transferase activity preferentially on single-strand DNA (ssDNA) and double-strand DNA containing 3’ ssDNA overhangs, referred to as partial ssDNA (pssDNA) (Kent et al., 2016). This study also compared the terminal transferase activities of Polθ and terminal deoxynucleotidyl transferase (TdT) using their respective optimal conditions, and found that Polθ is a more versatile enzyme for modifying the 3’ terminus of nucleic acids. For example, the authors showed that Polθ is able to modify nucleic acids with a wider variety of nucleotide analogs, such as those containing large fluorophores or attachment chemistries (Kent et al., 2016). As a specific example, Polθ was shown to efficiently modify ssDNA with a nucleotide analog containing click chemistry applicability (i.e., a linker attached to an azide group), whereas TdT failed to use the same nucleotide as a substrate (Kent et al., 2016). TdT was also unable to use a Texas Red conjugated nucleotide analog that Polθ efficiently utilized to modify ssDNA (Kent et al., 2016). Polθ is also capable of modifying the 3’ terminal ends of RNA and appears to show a significantly lower discrimination against ribonucleotides compared to TdT (Kent et al., 2016). Altogether, this recent report demonstrates that Polθ is a more versatile terminal transferase enzyme than TdT and therefore should be more useful for a wide range of applications in biotechnology and biomedical research that require modification of 3’ terminal DNA and RNA ends (Kent et al., 2016). Here, we explain in detail step-by-step procedures for using Polθ as a robust terminal transferase enzyme in vitro.

Materials and Reagents

  1. The following reagents are needed for modifying nucleic acids with Polθ:
    1. Pipette tips (i.e., Fisher Scientific, catalog number: 02-707-432 )
    2. Microcentrifuge tubes (i.e., 0.5 ml or 1.5 ml). DNase and RNase free tubes are recommended for reactions (i.e., BioDot Ultra Spin 1.5 ml Microcentrifuge Tubes) (DOT Scientific, catalog number: 711-FTG )
    3. ssDNA or RNA to be modified (typically 10-50 nt in length; desalted, HPLC or PAGE purified)
    4. Purified Polθ (residues 1,792-2,590, MW = 90 kDa) (expression vector and purification methods: Hogg et al., 2011)
    5. Nucleoside triphosphate analogs (i.e., TriLink BioTechnologies, catalog number: N-2008-102502 and TriLink BioTechnologies, catalog number: N-5001 ) or canonical nucleoside triphosphates (i.e., Promega, catalog number: U120 )
    6. 1 M Tris buffer pH 8.2 (i.e., DOT Scientific, catalog number: DST60040-10000 )
    7. Hydrochloric acid (HCl) (i.e., Thomas Scientific, catalog number: C395L46 )
    8. NP-40 detergent (i.e., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28324 )
    9. Bovine serum albumin (BSA) (protease free) (i.e., Fisher Scientific, catalog number: BP9703100 )
    10. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (i.e., Sigma-Aldrich, catalog number: M3634-100G )
    11. 1 M HEPES buffer pH 8.0 (i.e., Oakwood Products, catalog number: 047861-1Kg )
    12. Sodium hydroxide (NaOH)
    13. Deionized water (dH2O) (Autoclaved Nanopure filtered water is recommended for reactions with RNA)
    14. 1 M Tris buffer pH 8.2 (see Recipes)
    15. Buffer A (see Recipes)
    16. 1 M HEPES buffer pH 8.0 (see Recipes)

  2. The following reagents are needed if visualization of RNA and DNA modification is desired: 
    1. Ammonium persulfate (APS) (i.e., Sigma-Aldrich, catalog number: A3678 )
    2. 40% acrylamide solution (19:1 acrylamide:bis acrylamide) (i.e., Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9022 )
    3. Ethylenediaminetetraacetic acid (EDTA) (i.e., Sigma-Aldrich, catalog number: 03620 )
    4. Tetramethylethylenediamine (TEMED) (i.e., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17919 )
    5. Formamide (i.e., Sigma-Aldrich)
    6. 1 M HEPES buffer pH 8.0 (i.e., Oakwood Products, catalog number: 047861-1Kg )
    7. Sodium chloride (NaCl) (i.e., Sigma-Aldrich, catalog number: S9888 )
    8. Glycerol (i.e., Avantor Performance Materials, Macron Fine Chemicals, catalog number: 5092-16 )
    9. NP-40 detergent (i.e., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28324 )
    10. Xylene cyanol (i.e., Fischer Scientific, catalog number: BP125-100 )
    11. Bromophenol blue (i.e., DOT Scientific, catalog number: DSB40160-25 )
    12. Dithiothreitol (DTT) (i.e., bioWORLD, catalog number: 40400120 )
    13. Buffer B (see Recipes)
    14. 2x stop buffer (see Recipes)

Equipment

  1. The following equipment is needed for modifying nucleic acids with Polθ:
    1. Pipettes (i.e., P2, P20)
    2. Temperature controlled water bath or incubator

  2. The following equipment is needed if visualization of RNA and DNA modification is desired:
    1. Large vertical sequencing gel apparatus (i.e., APOGEE ELECTROPHORESIS, model: Model S2 ) or small vertical gel apparatus (i.e., Bio-Rad Laboratories)
    2. Glass plates for large or small gels
      Note: Large glass plates for APOGEE ELECTROPHORESIS Model S2 SEQUENCER can be obtained from APOGEE ELECTROPHORESIS, and standard small plates and apparatuses can be obtained from Bio-Rad Laboratories.
    3. Plastic combs for gels
      Note: Plastic combs for large sequencing gels can be obtained from APOGEE ELECTROPHORESIS.
    4. Gel spacers 0.4 mm thick (i.e., APOGEE ELECTROPHORESIS)
    5. Electrophoresis power supply (standard voltage [i.e., 300 V] source for small gels, high voltage [i.e., 5,000 V] source for large sequencing gels)
      Note: Power supplies may be obtained from Bio-Rad Laboratories.
    6. Fluorescence imaging system (for fluorophore labeled nucleic acids)
    7. Film developer or phosphorimager (i.e., FUJIFILM, model: FLA7000 ) (for 5’ 32P radio-labeled nucleic acids)

Procedure

  1. Modification of the 3’ terminal ends of DNA and RNA using Polθ
    1. Purified Polθ is needed for modifying the 3’ terminal ends of DNA and RNA. Procedures for expressing and purifying Polθ from E. coli are described in previous studies (Hogg et al., 2011).

      Notes:

      1. Synthetic single-stranded DNA (ssDNA) and RNA typically ~10-50 nt (nucleotides) in length have been routinely modified in our laboratory. Thus, we recommend using nucleic acids of similar length for the following procedure.
      2. All reagents below are listed as final concentrations.
    2. A typical procedure for modifying ssDNA or RNA is described as follows:
      1. 50-100 nM of the ssDNA or RNA to be modified is mixed with 50-500 μM concentration of the desired nucleotide used for modification in buffer A (see Recipes) along with 5 mM MnCl2 in a reaction volume of 10-20 μl.
        Note: We have not thoroughly tested the effects of different nucleotide concentrations. However, concentration ranges between 50-500 μM have shown efficient terminal transferase activity. Most of our previous reactions included 500 μM nucleotides. Reaction volumes can be varied according to preference and concentrations of ssDNA and RNA used successfully in the reaction in our experience are 50-100 nM. Importantly, the DNA or RNA must include a 3’ terminal nucleotide containing a hydroxyl group at the 3’ position of the sugar moiety for Polθ terminal transferase activity to occur.
      2. The terminal transferase reaction is then initiated by adding 200-500 nM purified Polθ and incubating at 42 °C for 2 h. Reactions are gently mixed with a pipette. Vortexing is not recommended.
      3. Reactions can be terminated by heating to ≥ 80 °C for 10 min or by the addition of ≥ 10 mM EDTA. Although the amount of Polθ can vary, optimal terminal transferase activity is observed with a 5-10 higher ratio of polymerase to nucleic acid molecule. We note that relatively high concentrations (i.e., > 50 mM) of salt (e.g., NaCl) suppress Polθ terminal transferase activity.
    3. The 2 h incubation time specified in the above procedure will give rise to multiple (i.e., 3 to > 100) terminal transfer events for most canonical nucleotides. However, in some cases only a single transfer event may occur depending on the particular nucleotide analog used. For example, certain nucleotide analogs may not be efficiently incorporated by Polθ and thus limit the enzyme to a single nucleotide transfer event. For determining the number of terminal transferase events that occur on a given substrate in the presence of particular nucleotides, we recommend visualizing the initial nucleic acid substrate and nucleic acid reaction products in a denaturing sequencing gel as described below in the Data analysis section.

  2. Examples of experimental procedures for modifying the 3’ terminal ends of DNA and RNA using Polθ
    1. As examples of Polθ terminal transferase activity on ssDNA and RNA, reactions were performed as follows. 50 nM of 5’ 32P radio-labeled ssDNA oligo (sequence indicated; Figures 1A and 1B) or RNA (sequence indicated; Figure 1C) was mixed with 50 µM (Figures 1A and 1B) or 500 µM.


      Figure 1. Use of Polθ terminal transferase activity to modify the 3’ terminal ends of DNA and RNA. A-C. Denaturing gels showing Polθ terminal transferase activity on the indicated ssDNA (A and B) and RNA (C) substrates in the presence of the indicated nucleotides. Lanes 1 lack Polθ and nucleotides. Lanes 2 and 3 include Polθ and the indicated nucleotides. ssDNA and RNA sequences are indicated at bottom. *, 32P radio-label.

    2. (Figure 1C) of the indicated nucleotides along with 5 mM MnCl2 in 20 µl of buffer A (see Recipes).
    3. Reactions were initiated by adding 200 nM of purified Polθ (stored in buffer B [see Recipes]), then incubating at 42 °C.
    4. After 2 h, reactions were terminated by adding 20 µl of 2x stop buffer (see Recipes).
    5. Radio-labeled ssDNA and RNA were then analyzed after denaturing gel electrophoresis and autoradiography as described below in the Data analysis section. The data show that Polθ efficiently transfers nucleotides to the 3’ terminus of nucleic acid substrates as demonstrated in previous studies (Figure 1) (Kent et al., 2016). These experiments also demonstrate the ability of Polθ to efficiently transfer large nucleotide analogs, consistent with recent work (Kent et al., 2016). We note that Polθ may also be used to modify double-strand blunt ended DNA, however, fewer nucleotides are transferred to these substrates as demonstrated in previous work (Kent et al., 2016). Partial single-strand DNA containing 3’ overhangs are most efficiently extended by Polθ (Kent et al., 2016).

Data analysis

  1. Visualizing modified RNA and DNA in denaturing gels
    1. Reactions should be performed as above with the following modifications. The RNA or DNA should be either 5’ radio-labeled, or conjugated with a fluorophore prior to the reaction for their detection in denaturing gels. Nucleic acids can be radio-labeled using T4 polynucleotide kinase in the presence of gamma-ATP. For fluorophore detection, DNA and RNA oligonucleotides can be purchased with 5’ fluorophore linkages. We recommend terminating reactions with an equal volume of 2x stop buffer (see Recipes).
    2. Initial nucleic acid substrates and reaction products should be resolved in standard urea denaturing 10-20% polyacrylamide gels. Helpful protocols for pouring and processing urea denaturing polyacrylamide sequencing gels are referenced here (Summer et al., 2009; Flett, et al., 2013). Large sequencing gels will allow for the highest resolution (i.e., single nucleotide resolution). However, smaller gels may provide enough resolution depending on the particular application. In the case of RNA, we recommend adding 10-15% formamide to urea denaturing polyacrylamide gels to reduce RNA secondary structures that can appear as smears in the gel. Large sequencing gels are typically run at 70-80 W using a high voltage (5,000 V) power supply. The resolved nucleic acids can then be visualized using a fluorescent imager (for 5’ fluorophore conjugated oligos) or using a phosphorimager or autoradiography (for 5’ 32P labeled oligos). Figure 1 shows examples of 5’ 32P radio-labeled nucleic acids that were resolved in large sequencing gels, then visualized by autoradiography.

Notes

  1. Reproducibility
    In our experience, extension of nucleic acids by Polθ is highly reproducible. However, we note that the precise amount of initial substrates extended may vary. For example, in some cases 100% of nucleic acid substrates are extended, whereas in other cases a small fraction (i.e., ~5-15%) of substrates are not extended. A 4-5 fold higher ratio of Polθ to nucleic acid substrates will usually allow for the majority of substrates to be extended. We note that the number of nucleotides transferred to the 3’ terminus of nucleic acids may vary. Thus, the final length of extended nucleic acids will not be identical for all molecules. The respective structures of canonical nucleotides and nucleotide analogs will also give rise to different terminal transferase efficiencies. For example, deoxyadenosine monophosphate is most efficiently transferred by Polθ (Kent et al., 2016). Other deoxyribonucleotides are somewhat less efficiently transferred by the polymerase (Kent et al., 2016). The initial nucleic acid sequence may also affect Polθ terminal transferase activity. Previous studies compare the efficiency of Polθ terminal transferase activity on different nucleic acid substrates and in the presence of various canonical nucleotides and nucleotide analogs (Kent et al., 2016).
  2. Additional notes, technical tips and cautionary points
    For optimal Polθ terminal transferase activity, we recommend storing the enzyme in buffer B (see Recipes) at concentrations ≥ 1 mg/ml in small aliquots at -80 °C and limiting freeze thaw cycles to 2-3 times. We note that oligonucleotides relatively short in length (< 10 nt) may not be extended as efficiently as those longer in length (> 10 nt). Oligos containing a high proportion of closely spaced guanosine bases, for example similar to telomere repetitive DNA sequences or those that form G quadruplexes, may exhibit a lower efficiency of extension by Polθ (Kent et al., 2016). As noted above, Polθ can also be used to modify double-stranded DNA, however, only 1-3 nucleotides are generally transferred to these substrates (Kent et al., 2016).

Recipes

  1. 1 M Tris buffer pH 8.2
    Weigh 121.1 g of Tris Ultra Pure and add 800 ml of dH2O
    Stir until dissolved, then adjust pH to 8.2 with HCl
    Adjust final volume to 1 L with dH2O
  2. Buffer A
    20 mM Tris-HCl pH 8.2
    0.01% NP-40
    0.1 mg/ml BSA
    10% glycerol
  3. 1 M HEPES buffer pH 8.0
    Weigh 238.3 g of HEPES and add 800 ml of dH2O
    Stir until dissolved, then adjust pH to 8.0 with NaOH
    Adjust final volume to 1 L with dH2O
  4. Buffer B
    50 mM HEPES pH 8.0
    300 mM NaCl
    10% glycerol
    0.01% NP-40
    5 mM DTT
  5. 2x stop buffer
    90% formamide
    50 mM EDTA
    0.03% xylene cyanol
    0.03% bromophenol blue

Acknowledgments

This work was funded by National Institutes of Health grant 1R01GM115472-01 awarded to R.T.P. Competing interests: R.T.P. and T.K. filed a patent application about the use of DNA polymerase theta to modify the 3’ terminus of nucleic acids. The protocol described herein was adapted from previous studies (Kent et al., 2016).

References

  1. Black, S. J., Kashkina, E., Kent, T. and Pomerantz, R. T. (2016). DNA polymerase theta: A unique multifunctional end-joining machine. Genes (Basel) 7(9).
  2. Chan, S. H., Yu, A. M. and McVey, M. (2010). Dual roles for DNA polymerase theta in alternative end-joining repair of double-strand breaks in Drosophila. PLoS Genet 6(7): e1001005.
  3. Flett, F. and Interthal, H. (2013). Separation of DNA oligonucleotides using denaturing urea PAGE. Methods Mol Biol 1054: 173-185.
  4. Hogg, M., Seki, M., Wood, R. D., Doublie, S. and Wallace, S. S. (2011). Lesion bypass activity of DNA polymerase theta (POLQ) is an intrinsic property of the pol domain and depends on unique sequence inserts. J Mol Biol 405(3): 642-652.
  5. Kent, T., Chandramouly, G., McDevitt, S. M., Ozdemir, A. Y. and Pomerantz, R. T. (2015). Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase theta. Nat Struct Mol Biol 22(3): 230-237.
  6. Kent, T., Mateos-Gomez, P. A., Sfeir, A. and Pomerantz, R. T. (2016). Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining. Elife 5.
  7. Koole, W., van Schendel, R., Karambelas, A. E., van Heteren, J. T., Okihara, K. L. and Tijsterman, M. (2014). A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites. Nat Commun 5: 3216.
  8. Mateos-Gomez, P. A., Gong, F., Nair, N., Miller, K. M., Lazzerini-Denchi, E. and Sfeir, A. (2015). Mammalian polymerase theta promotes alternative NHEJ and suppresses recombination. Nature 518(7538): 254-257.
  9. Sfeir, A. and Symington, L. S. (2015). Microhomology-mediated end joining: A back-up survival mechanism or dedicated pathway? Trends Biochem Sci 40(11): 701-714.
  10. Summer, H., Gramer, R. and Droge, P. (2009). Denaturing URea polyacrylamide gel electrophoresis (Urea PAGE). J Vis Exp (32): 1485.
  11. Wood, R. D. and Doublie, S. (2016). DNA polymerase theta (POLQ), double-strand break repair, and cancer. DNA Repair (Amst) 44: 22-32.
  12. Yousefzadeh, M. J., Wyatt, D. W., Takata, K., Mu, Y., Hensley, S. C., Tomida, J., Bylund, G. O., Doublie, S., Johansson, E., Ramsden, D. A., McBride, K. M. and Wood, R. D. (2014). Mechanism of suppression of chromosomal instability by DNA polymerase POLQ. PLoS Genet 10(10): e1004654.

简介

DNA聚合酶θ(Polθ)是一种混杂的酶,对易错的DNA双链断裂(DSB)修复途径而言是必需的,称为替代性末端连接(alt-EJ)。 在这种形式的DSB修复中,Polθ通过模板和非模板核苷酸添加循环在切割的DSB的3'末端处进行末端转移酶活性。 由于人Polθ能够用广泛的多种核糖核苷酸和脱氧核糖核苷酸类似物修饰DNA和RNA的3'末端,因此其末端转移酶活性对于生物技术应用比末端脱氧核苷酸转移酶(TdT)更有用。 在这里,我们详细介绍使用纯化的人Polθ修饰生物技术和生物医学研究中各种应用的RNA和DNA的3'末端的简单方法。
【背景】人类POLQ基因编码含有N末端超家族2(SF2)型解旋酶结构域和C末端A家族聚合酶结构域的大蛋白质(Sfeir和Symington,2015; Black et al。,2016; Wood and Doublie, 2016)。蛋白质也编码一个大的中心结构域,其功能尚未归入。 Polθ在后生动物中表达,已被证明在DNA复制和修复的多个方面起作用(Black et al。,2016; Wood and Doublie,2016)。最近的工作表明,哺乳动物Polθ对于易于识别的DNA双链断裂(DSB)修复途径而言是必需的,称为替代性末端连接(alt-EJ),也称微小鼠介导的终结合(MMEJ)(Yousefzadeh et al。 ,2014; Kent等人,2015; Mateos-Gomez等人,2015)。聚合酶的这种基本功能在后生动物中是保守的(Chan et al。,2010; Koole et al。,2014)。
有趣的是,与更准确的非同源末端连接(NHEJ)途径相比,Polθ介导的alt-EJ导致在DNA修复连接处相对较大的缺失和插入(indel)(Black等,2016)。例如,alt-EJ通常产生1-6个碱基对(bp)的插入,在某些情况下插入可能超过30bp(Yousefzadeh等,2014; Mateos-Gomez等,2015; Black et al。 ,2016; Kent等,2016)。有趣的是,来自无脊椎动物和脊椎动物系统的多项研究表明,一些插入区是通过附近的DNA序列进行模板化的,如DSB侧翼(Black等,2016)。在其他情况下,插入序列似乎是随机的(Black et al。,2016)。这些和其他研究导致Polθ可能通过模板和非模板末端转移酶机制在DSB中产生插入片段的想法。
事实上,在最近的一项研究中,Kent等证明人Polθ聚合酶结构域(以下称为Polθ)优先于单链DNA(ssDNA)和含有3'ssDNA突出端的双链DNA(称为部分ssDNA(pssDNA))(Kent et等等,2016)。本研究还使用其各自的最佳条件比较了Polθ和末端脱氧核苷酸转移酶(TdT)的末端转移酶活性,发现Polθ是修饰核酸3'端的更通用的酶。例如,作者表明,Polθ能够用更多种类的核苷酸类似物修饰核酸,例如含有大量荧光团或附着化学物质的核酸(Kent等,2016)。作为具体实例,Polθ被证明可以用含有点击化学适用性的核苷酸类似物(即,连接到叠氮基的连接体)有效地修饰ssDNA,而TdT不能使用与底物相同的核苷酸(Kent等,2016 )。 TdT也不能使用Polθ有效利用来修饰ssDNA的德克萨斯红缀合核苷酸类似物(Kent等,2016)。与TdT相比,Polθ还能够修饰RNA的3'末端并且似乎显示出比核糖核苷酸显着更低的鉴别(Kent等,2016)。总之,最近的报告显示,Polθ是一种比TdT更为通用的末端转移酶,因此应用于需要修饰3'末端DNA和RNA末端的生物技术和生物医学研究中的广泛应用中更有用(Kent et al。 ,2016)。在这里,我们详细解释在体外使用Polθ作为稳定的末端转移酶的逐步程序。

关键字:DNA聚合酶, DNA修复, DNA修饰, 替代末端连接, 末端脱氧核苷酸转移酶, 生物技术, 核苷酸类似物

材料和试剂

  1. 用Polθ修饰核酸需要以下试剂:
    1. 移液器提示(即,Fisher Scientific,目录号:02-707-432)
    2. 微量离心管(即,即0.5ml或1.5ml)。推荐使用无DNA酶和无RNA酶的反应管(即BioDot Ultra Spin 1.5 ml微量离心管)(DOT Scientific,目录号:711-FTG)
    3. ssDNA或RNA(通常长度为10-50nt;脱盐,HPLC或PAGE纯化)
    4. 纯化的Polθ(残基1,792-2,590,MW = 90kDa)(表达载体和纯化方法:Hogg等人,2011)
    5. 核苷三磷酸类似物(例如,TriLink BioTechnologies,目录号:N-2008-102502和TriLink BioTechnologies,目录号:N-5001)或典型的核苷三磷酸(例如 Promega,目录号:U120)
    6. 1M Tris缓冲液pH 8.2(即,DOT Scientific,目录号:DST60040-10000)
    7. 盐酸(HCl)(即,Thomas Scientific,目录号:C395L46)
    8. NP-40洗涤剂(即Thermo Fisher Scientific,Thermo Scientific TM,目录号:28324)
    9. 牛血清白蛋白(BSA)(不含蛋白酶)(即,Fisher Scientific,目录号:BP9703100)
    10. 二氯化锰(II)四水合物(MnCl 2·4H 2 O)(Sigma-Aldrich,目录号:M3634-100G)
    11. 1 M HEPES缓冲液pH 8.0(即,Oakwood Products,目录号:047861-1Kg)
    12. 氢氧化钠(NaOH)
    13. 去离子水(dH 2 O)(推荐用于与RNA反应的高压灭菌的纳米级过滤水)
    14. 1 M Tris缓冲液pH 8.2(参见食谱)
    15. 缓冲液A(参见食谱)
    16. 1 M HEPES缓冲液pH 8.0(见配方)

  2. 如果需要进行RNA和DNA修饰的可视化,则需要以下试剂: 
    1. 过硫酸铵(APS)(即,Sigma-Aldrich,目录号:A3678)
    2. 40%丙烯酰胺溶液(19:1丙烯酰胺:双丙烯酰胺)(即Thermo Fisher Scientific,Invitrogen,Supest TM,目录号:AM9022)
    3. 乙二胺四乙酸(EDTA)(即,Sigma-Aldrich,目录号:03620)
    4. 四甲基乙二胺(TEMED)(即Thermo Fisher Scientific,Thermo Scientific TM,目录号:17919)
    5. 甲酰胺(即,Sigma-Aldrich)
    6. 1 M HEPES缓冲液pH 8.0(即,Oakwood Products,目录号:047861-1Kg)
    7. 氯化钠(NaCl)(即,Sigma-Aldrich,目录号:S9888)
    8. 甘油(即,Avantor Performance Materials,Macron Fine Chemicals,目录号:5092-16)
    9. NP-40洗涤剂(即Thermo Fisher Scientific,Thermo Scientific TM,目录号:28324)
    10. 二甲苯胞醇(即,Fischer Scientific,目录号:BP125-100)
    11. 溴酚蓝(,DOT Scientific,目录号:DSB40160-25)
    12. 二硫苏糖醇(DTT)(即,,bioWORLD,目录号:40400120)
    13. 缓冲液B(参见食谱)
    14. 2x停止缓冲区(见配方)

设备

  1. 用Polθ修饰核酸需要以下设备:
    1. 移液器(即,P2,P20)
    2. 温控水浴或孵化器

  2. 如果需要RNA和DNA修饰的可视化,则需要以下设备:
    1. 大型垂直测序凝胶装置(即,APOGEE ELECTROPHORESIS,型号:S2型)或小型垂直凝胶装置(即,Bio-Rad Laboratories)
    2. 用于大或小凝胶的玻璃板
      注意:APOGEE ELECTROPHORESIS S2型序列的大玻璃板可以从APOGEE ELECTROPHORESIS获得,标准的小型板和设备可以从Bio-Rad Laboratories获得。
    3. 塑胶梳子用于凝胶
      注意:可以从APOGEE ELECTROPHORESIS获得大型测序凝胶的塑料梳。
    4. 凝胶间隔物0.4mm厚(即,APOGEE ELECTROPHORESIS)
    5. 电泳电源(小型凝胶的标准电压(即,300 V)源,大型测序凝胶的高电压[...],5,000 V]源) > 注意:电源可能来自Bio-Rad实验室。
    6. 荧光成像系统(用于荧光团标记的核酸)
    7. 薄膜显影剂或荧光成像仪(即,FUJIFILM,型号:FLA7000)(对于5'32P放射性标记的核酸)

程序

  1. 使用Polθ修饰DNA和RNA的3'末端
    1. 需要纯化的Polθ来修饰DNA和RNA的3'末端。用于表达和纯化来自E的Polθ的程序。以前的研究(Hogg等人,2011)中描述了大肠杆菌。

      注意:

      1. 通常在我们的实验室中常规地修改长度为约10-50nt(核苷酸)的合成单链DNA(ssDNA)和RNA。因此,我们建议使用类似长度的核酸进行以下程序。
      2. 以下所有试剂均列为最终浓度。
    2. 修饰ssDNA或RNA的典型方法描述如下:
      1. 将50-100nM的待修饰的ssDNA或RNA与50-500μM浓度的用于缓冲液A中修饰的所需核苷酸(参见食谱)和5mM MnCl 2反应混合体积10-20μl。
        注意:我们尚未彻底测试不同核苷酸浓度的影响。然而,浓度范围在50-500μM之间已经显示出有效的末端转移酶活性。我们以前的大多数反应包括500μM核苷酸。反应体积可以根据我们的经验中在反应中成功使用的ssDNA和RNA的优选和浓度而变化,为50-100nM。重要的是,DNA或RNA必须包含在糖部分的3'位置含有羟基的3'末端核苷酸,用于Polθ末端转移酶活性的发生。
      2. 然后通过加入200-500nM纯化的Polθ并在42℃下孵育2小时来引发末端转移酶反应。反应与移液管轻轻混合。不推荐旋转。
      3. 反应可以通过加热至≥80℃10分钟或通过加入≥10mMEDTA来终止。尽管Polθ的量可以变化,但观察到最佳的末端转移酶活性,聚合酶与核酸分子的比例高达5-10倍。我们注意到相对高的浓度(即,> 50mM)的盐(例如NaCl)抑制Polθ末端转移酶活性。
    3. 在上述程序中规定的2小时孵育时间将导致大多数规范核苷酸的多个(即,3至> 100)末端转移事件。然而,在某些情况下,根据所使用的特定核苷酸类似物,仅可能发生单次转移事件。例如,某些核苷酸类似物可能无法通过Polθ有效并入,从而将酶限制为单核苷酸转移事件。为了确定在特定核苷酸存在下在给定底物上发生的末端转移酶事件的数量,我们建议在变性测序凝胶中显现初始核酸底物和核酸反应产物,如下面在数据分析部分所述。 />
  2. 使用Polθ修饰DNA和RNA的3'末端的实验步骤的实例
    1. 作为对ssDNA和RNA的Polθ末端转移酶活性的实例,如下进行反应。将50nM的5'末端p放射性标记的ssDNA寡核苷酸(序列表示;图1A和1B)或RNA(序列指示;图1C)与50μM(图1A和1B)或500 μM。


      图1.使用Polθ末端转移酶活性修饰DNA和RNA的3'末端。 A-C。在指定的核苷酸存在下,在指定的ssDNA(A和B)和RNA(C))底物上显示出Polθ末端转移酶活性的变性凝胶。泳道1缺少Polθ和核苷酸。泳道2和3包括Polθ和指定的核苷酸。 ssDNA和RNA序列显示在底部。 *, 32 P无线电标签。

    2. (图1C)以及20μl缓冲液A中的5mM MnCl 2(参见食谱)。
    3. 通过加入200nM纯化的Polθ(储存在缓冲液B中(参见食谱))开始反应,然后在42℃下孵育。
    4. 2小时后,加入20μl2x停止缓冲液终止反应(参见食谱)
    5. 然后如下面的数据分析部分所述,在变性凝胶电泳和放射自显影之后分析放射性标记的ssDNA和RNA。数据显示,Polθ有效地将核苷酸转移到核酸底物的3'末端,如先前的研究(图1)所示(Kent等人,2016)。这些实验还证明了Polθ有效转移大核苷酸类似物的能力,与最近的工作一致(Kent等人,2016)。我们注意到,Polθ也可以用于修饰双链平端DNA,然而,如先前的研究(Kent等人,2016)所证明,较少的核苷酸被转移到这些底物。含有3'突出端的部分单链DNA最有效地被Polθ延伸(Kent等人,2016)。

数据分析

  1. 在变性凝胶中可视化修饰的RNA和DNA
    1. 反应应如上所述进行,并进行以下修改。反应前RNA或DNA应该是5'放射性标记的,或与荧光团缀合,以便在变性凝胶中进行检测。在γ-ATP存在下,核酸可以使用T4多核苷酸激酶进行放射标记。对于荧光团检测,可以用5'荧光团连接购买DNA和RNA寡核苷酸。我们建议用等量的2x停止缓冲液终止反应(见配方)。
    2. 初始核酸底物和反应产物应在标准尿素变性10-20%聚丙烯酰胺凝胶中解析。在此引用用于倾倒和处理尿素变性聚丙烯酰胺测序凝胶的有用方案(Summer& et al。,2009; Flett,et al。,2013)。大的测序凝胶将允许最高分辨率(,即单核苷酸分辨率)。然而,根据具体应用,较小的凝胶可提供足够的分辨率。在RNA的情况下,我们建议在尿素变性聚丙烯酰胺凝胶中加入10-15%的甲酰胺,以减少可能在凝胶中显示为污迹的RNA二级结构。大型测序凝胶通常使用高电压(5,000 V)电源运行在70-80 W。然后可以使用荧光成像仪(用于5'荧光团缀合的寡聚体)或使用磷光体仪或放射自显影术(对于5',32位标记的寡核苷酸)可视化解析的核酸。图1显示了在大型测序凝胶中解析的5'末端32放射性标记的核酸的实例,然后通过放射自显影进行显像。

笔记

  1. 重复性
    在我们的经验中,Polθ延伸核酸是高度可重复的。然而,我们注意到扩展的初始底物的精确量可能会有所不同。例如,在一些情况下,100%的核酸底物延伸,而在其他情况下,小片段(即,约5-15%)的底物不延长。 Polθ与核酸底物的比例高4-5倍通常允许大部分底物延伸。我们注意到转移到核酸的3'末端的核苷酸数目可能有所不同。因此,所有分子的延伸核酸的最终长度将不相同。典型核苷酸和核苷酸类似物的各自结构也将产生不同的末端转移酶效率。例如,脱氧腺苷一磷酸通过Polθ(Kent等人,2016)最有效地转移。其他脱氧核糖核苷酸通过聚合酶稍微有效地转移(Kent等人,2016)。初始核酸序列也可能影响Polθ末端转移酶活性。以前的研究比较了Polθ末端转移酶活性对不同核酸底物的效率和各种规范核苷酸和核苷酸类似物的存在(Kent等人,2016)。
  2. 附加说明,技术提示和警戒点
    为了获得最佳的Polθ末端转移酶活性,我们建议在-80°C的小等分试样中将酶以浓度≥1 mg/ml的缓冲液B(见配方)存放,并将冻融循环限制在2-3次。我们注意到,长度相对较短(<10nt)的寡核苷酸可能不会像那些长度较长(> 10nt)的那样有效地延长。寡核苷酸含有高比例的紧密间隔的鸟苷碱基,例如类似于端粒重复DNA序列或形成G四链体的序列,可能表现出较低的Polθ延伸效率(Kent等,2016) 。如上所述,Polθ也可以用于修饰双链DNA,但是,通常仅将1-3个核苷酸转移到这些底物(Kent等人,2016)。

食谱

  1. 1 M Tris缓冲液pH 8.2
    称取121.1g Tris Ultra Pure,并加入800ml dH 2 O-/ 搅拌至溶解,然后用盐酸调节pH至8.2 将最终体积调整为1 L,dH <2> O
  2. 缓冲区A
    20mM Tris-HCl pH 8.2
    0.01%NP-40
    0.1 mg/ml BSA
    10%甘油
  3. 1 M HEPES缓冲液pH 8.0
    称取238.3g HEPES并加入800ml dH 2 O-/ 搅拌直至溶解,然后用NaOH调节pH至8.0 将最终体积调整为1 L,dH <2> O
  4. 缓冲区B
    50 mM HEPES pH 8.0
    300 mM NaCl
    10%甘油
    0.01%NP-40
    5 mM DTT
  5. 2x停止缓冲区
    90%甲酰胺
    50 mM EDTA
    0.03%二甲苯甲醇
    0.03%溴酚蓝

致谢

这项工作由美国国家卫生研究院资助,授予R.T.P.授予1R01GM115472-01。竞争兴趣:R.T.P.和T.K.提交了关于使用DNA聚合酶θ修饰核酸的3'末端的专利申请。本文所述的方案改编自以前的研究(Kent等人,2016)。

参考

  1. Black,SJ,Kashkina,E.,Kent,T.and Pomerantz,RT(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27657134"target ="_ blank"> DNA polymerase theta:一种独特的多功能终端连接机。基因(巴塞尔) 7(9)。
  2. Chan,SH,Yu,AM和McVey,M。(2010)。  DNA聚合酶的双重角色在果蝇中双链断裂的替代性末端连接修复中。 PLoS Genet 6(7) :e1001005。
  3. Flett,F.和Interthal,H。(2013)。  使用变性尿素PAGE分离DNA寡核苷酸方法Mol Biol 1054:173-185。
  4. Hogg,M.,Seki,M.,Wood,RD,Doublie,S。和Wallace,SS(2011)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm。 nih.gov/pubmed/21050863"target ="_ blank"> DNA聚合酶(POLQ)的病变旁路活性是pol结构域的固有特性,取决于独特的序列插入物。/em> 405(3):642-652。
  5. Kent,T.,Chandramouly,G.,McDevitt,SM,Ozdemir,AY and Pomerantz,RT(2015)。 
  6. Kent,T.,Mateos-Gomez,PA,Sfeir,A.and Pomerantz,RT(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27311885"target ="_ blank">聚合酶θ是一种稳定的末端转移酶,其在终止连接期间在三种不同机制之间振荡。 5.
  7. Koole,W.,van Schendel,R.,Karambelas,AE,van Heteren,JT,Okihara,KL和Tijsterman,M。(2014)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/24496117"target ="_ blank">聚合酶依赖性修复途径抑制内源性G4 DNA位点广泛的基因组不稳定性。 5:3216.
  8. Mateos-Gomez,PA,Gong,F.,Nair,N.,Miller,KM,Lazzerini-Denchi,E。和Sfeir,A.(2015)。哺乳动物聚合酶θ促进替代NHEJ并抑制重组。自然 518(7538): 254-257。
  9. Sfeir,A.和Symington,LS(2015)。微生物学介导的终端加入:后备生存机制或专用途径?趋势生物化学 40(11):701-714。
  10. Summer,H.,Gramer,R.and Droge,P。(2009)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/19865070" 目标="_ blank">变性URea聚丙烯酰胺凝胶电泳(尿素PAGE)。 Vis Vis (32):1485.
  11. Wood,RD和Doublie,S.(2016)。  DNA聚合酶(POLQ),双链断裂修复和癌症。 DNA修复(Amst) 44:22-32。
  12. Yousefzadeh,MJ,Wyatt,DW,Takata,K.,Mu,Y.,Hensley,SC,Tomida,J.,Bylund,GO,Doublie,S.,Johansson,E。,Ramsden,DA,McBride,KM and Wood ,RD(2014)。  抑制染色体不稳定的机制 通过DNA聚合酶POLQ。 PLoS Genet 10(10):e1004654。
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Copyright Hoang et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Hoang, T. M., Kent, T. and Pomerantz, R. T. (2017). Modification of 3’ Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity. Bio-protocol 7(12): e2330. DOI: 10.21769/BioProtoc.2330.
  2. Kent, T., Mateos-Gomez, P. A., Sfeir, A. and Pomerantz, R. T. (2016). Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining. Elife 5.
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