Steady-state and Time-resolved Fluorescence Spectroscopy to Detect the Cruciform Structure in pBR322

doi:10.13560/j.cnki.biotech.bull.1985.2016.04.028

Abstract

Abstract: The specific aim of this work is to probe the formation process of DNA with cruciform extrusion in a pBR322 plasmid using spectroscopic methods while without digesting DNA into pieces. Pyrrolo deoxycitidine（PdC）was incorporated into pBR322 to substitute for dC at specific sites of 3 195，3 222 or 3 281. Steady-state and time-resolved fluorescence were employed to examine the specific properties of PdC in the cruciform region. Results were as followings：1）Steady-state fluorescent properties indicated that the fluorescence intensity of supercoiled PdC-pBR322 was about 4-fold stronger than that of relaxed PdC-pBR322，when PdC was incorporated into pBR322 at position 3 222；meanwhile，its time-resolved lifetime was about 0.3 ns longer than that of relaxed PdC-pBR3322. When PdC was incorporated at position 3 195 or 3 281，there was no significant change in either steady-state fluorescence spectra or time-resolved lifetime（about 1.42 ns at both positions）. 2）Lifetimes changed a little with the increasing of salt concentration，but not significantly. In conclusion，by analyzing fluorescence spectra and lifetimes（both in relaxed state and supercoiled plasmid），a cruciform structure with dC at site 3 222 of impaired loop is formed，and the cruciform is stable within this investigated salt concentration range（0-100 mmol/L）.

Key words: cruciform extrusion, pBR322 plasmid, PdC, steady-state and time-resolved fluorescence spectroscopy

XIA Lan， ,ZHANG Xu，. Steady-state and Time-resolved Fluorescence Spectroscopy to Detect the Cruciform Structure in pBR322[J]. Biotechnology Bulletin, 2016, 32(4): 210-216.

References

［1］高翼之. DNA双螺旋模型的建立——基因的物质本性［J］. 遗传, 2002, 24（6）：691-694.
［2］Qiu J, Liu J, Chen S, et al. Role of hairpin-quadruplex dna secondary structural conversion in the promoter of hnRNP K in gene transcriptional regulation［J］. Org Lett, 2015, 17（18）：4584-4587.
［3］Bevilacqua PC, Blose JM. Structures, kinetics, thermodynamics, and biological functions of RNA hairpins［J］. Annu Rev Phys Chem, 2008, 59：79-103.
［4］Chen J, Poddar NK, Tauzin LJ, et al. Single-molecule FRET studies of HIV TAR-DNA hairpin unfolding dynamics［J］. J Phys Chem B, 2014, 118（42）：12130-12139.
［5］Coufal J, Jagelská EB, Liao JC, et al. Preferential binding of p53 tumor suppressor to p21 promoter sites that contain inverted repeats capable of forming cruciform structure［J］. Biochem Biophys Res Commun, 2013, 441（1）：83-88.
［6］Li D, Lv B, Zhang H, et al. Positive supercoiling affiliated with nucleosome formation repairs non-B DNA structures［J］. Chem Commun（Camb）, 2014, 50（73）：10641-10644.
［7］赵宏宇, 蔡禄, 赵秀娟, 等. 化学药物对与人遗传病相关的DNA重复序列不稳定性的影响［J］. 生物技术通报, 2010（11）：153-156, 161.
［8］García-López A, Llamusí B, Orzáez M, et al. In vivo discovery of a peptide that prevents CUG-RNA hairpin formation and reverses RNA toxicity in myotonic dystrophy models［J］. Proc Natl Acad Sci USA, 2011, 108（29）：11866-11871.
［9］Pushechnikov A, Lee MM, Childs-Disney JL, et al. Rational design of ligands targeting triplet repeating transcripts that cause RNA dominant disease：application to myotonic muscular dystrophy type 1 and spinocerebellar ataxia type 3［J］. J Am Chem Soc, 2009, 131（28）：9767-9779.
［10］Wadkins RM, Vladu B, Tung CS, et al. Actinomycin D binds to metastable hairpins in single-stranded DNA［J］. Biochemistry, 1998, 37（34）：11915-11923.
［11］Wadkins RM, Tung CS, et al. The role of the loop in binding of an actinomycin D analog to hairpins formed by single-stranded DNA［J］. Arch Biochem Biophys, 2000, 384（1）：199-203.
［12］Jagelská EB, Pivonková H, Fojta M, et al. The potential of the cruciform structure formation as an important factor influencing p53 sequence-specific binding to natural DNA targets［J］. Biochem Biophys Res Commun, 2010, 391（3）：1409-1414.
［13］Shlyakhtenko LS, Hsieh P, et al. A cruciform structural transition provides a molecular switch for chromosome structure and dynamics［J］. J Mol Biol, 2000, 296（5）：1169-1173.
［14］Brázda V, Laister RC, Jagelská EB, et al. Cruciform structures are a common DNA feature important for regulating biological processes［J］. BMC Mol Biol, 2011, 12：33.
［15］Yahyaoui W, Callejo M, Price GB, et al. Deletion of the cruciform binding domain in CBP/14-3-3 displays reduced origin binding and initiation of DNA replication in budding yeast［J］. BMC Mol Biol, 2007, 8：27.
［16］Zannis-Hadjopoulos M, Yahyaoui W, Callejo M, et al. 14-3-3 cruciform-binding proteins as regulators of eukaryotic DNA replication［J］. Trends Biochem Sci, 2008, 33（1）：44-50.
［17］Hilton S, Winstanley D. Identification and functional analysis of the origins of DNA replication in the Cydia pomonella granulovirus genome［J］. J Gen Virol, 2007, 88（Pt 5）：1496-1504.
［18］Kim EL, Peng H, Esparza FM, et al. Cruciform-extruding regulatory element controls cell-specific activity of the tyrosine hydroxylase gene promoter［J］. Nucleic Acids Res, 1998, 26（7）：1793-1800.
［19］Horwitz MS. Transcription regulation in vitro by an E. coli promoter containing a DNA cruciform in the ‘-35’ region［J］. Nucleic Acids Res, 1989, 17（14）：5537-5545.
［20］Hanke JH, Hambor JE, Kavathas P, et al. Repetitive Alu elements form a cruciform structure that regulates the function of the human CD8 alpha T cell-specific enhancer［J］. J Mol Biol, 1995, 246（1）：63-73.
［21］Caffarelli E, Leoni L, Sampaolese B, et al. Persistence of cruciform structure and preferential location of nucleosomes on some regions of pBR322 and ColE 1 DNAs［J］. Eur J Biochem, 1986, 156（2）：335-342.
［22］Kato M, Hokabe S, Itakura S, et al. Interarm interaction of DNA cruciform forming at a short inverted repeat sequence［J］. Biophys J, 2003, 85（1）：402-408.
［23］张来斌, 任廷琦. 扩环荧光碱基类似物x-腺嘌呤分子基态和激发态性质的理论研究［J］. 物理学报, 2013, 10：353-359.
［24］Bhavsar YP, Reilly SM, Wadkins RM. Evaluation of fluorescent analogs of deoxycytidine for monitoring DNA transitions from duplex to functional structures［J］. J Nucleic Acids, 2011, 2011：986820.
［25］Tinsley RA, Walter NG. Pyrrolo-C as a fluorescent probe for monitoring RNA secondary structure formation［J］. RNA, 2006, 12（3）：522-529.
［26］Brázda V, Laister RC, Jagelská EB, et al. Cruciform structures are a common DNA feature important for regulating biological processes［J］. BMC Mol Biol, 2011, 12：33.
［27］ Stefanovsky VY, Moss T. The cruciform DNA mobility shift assay：A tool to study proteins that recognize bent DNA［J］. Methods Mol Biol, 2015, 1334：195-203.
［28］Brázda V, Jagelská EB, Liao JC, et al. The central region of BRCA1 binds preferentially to supercoiled DNA［J］. J Biomol Struct Dyn, 2009, 27（1）：97-104.
［29］Degtyareva N, Subramanian D, Griffith JD. Analysis of the binding of p53 to DNAs containing mismatched and bulged bases［J］. J Biol Chem, 2001, 276（12）：8778-8784.
［30］Jagelska EB, Pivonková H, Fojta M, et al. The potential of the cruciform structure formation as an important factor influencing p53 sequence-specific binding to natural DNA targets［J］. Biochem Biophys Res Commun, 2010, 391（3）：1409-1414.
［31］Zannis-Hadjopoulos M, Sibani S, Price GB. Eucaryotic replication origin binding proteins［J］. Front Biosci, 2004, 9：2133-2143.
［32］Kowalski D, Natale DA, Eddy MJ, et al. Stable DNA unwinding, not “breathing, ” accounts for single-strand-specific nuclease hypersensitivity of specific A+T-rich sequences［J］. Proc Natl Acad Sci U S A, 1988, 85（24）：9464-9468.