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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

Abstract

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability. 

Introduction

HTPP makes use of DNA binding microarray technology combined with biochemical analysis (Figure 1) to statistically identify specific features of DNA templates that affect the enzymatic activity of DNA primase. Therefore, HTPP provides a technological platform that facilitates a knowledge leap in the field. The classical tools used to determine primase recognition sites do not have the ability to yield massive amount of data, whereas HTPP does.

PBM is a technique routinely used to determine the binding preferences of transcription factors to DNA1,2; howe....

Protocol

1. Design of microarray

NOTE: DNA probes represent custom 36-nucleotide sequences, consisting of the recognition site for T7 DNA primase (GTC) located between two variable flanking regions, followed by a constant 24-nucleotide sequence tethered to a glass slide3. We used a 4 x 180,000 microarray format, which enabled spotting of each DNA sequence in six replicates, randomly distributed on the slide.

  1. Design of DNA library for primases with known recog.......

Representative Results

This technological advance for mapping the primase binding sites allows the obtaining of DNA binding properties that are difficult, if not impossible, to observe using classical tools. More importantly, HTPP enables the revisiting of the traditional understanding of primase binding sites. Specifically, HTPP reveals binding specificities in addition to known 5'-GTC-3' recognition sequences, which leads to changes in functional activities of T7 DNA primase. Namely, two groups of seq.......

Discussion

The PBM method has been widely used to investigate binding properties of transcription factors and can also be applied to DNA processing enzymes, such as DNA primase, that bind to DNA with low affinity. However, certain modifications of experimental procedures are required. The microarray experiment involves several steps: design of the DNA library, preparation of the chip, binding of the protein target, fluorescent labeling, and scanning. Mild washing steps are critical, since the long washes with solutions containing d.......

Acknowledgements

This research was supported by the ISRAEL SCIENCE FOUNDATION (grant no. 1023/18).

....

Materials

NameCompanyCatalog NumberComments
40% acrylamide-bisacrylamide (19:1) solutionMerck1006401000
95% formamideSigma-AldrichF9037-100ML
Alexa 488-conjugated anti-his antibodyQiagen35310
Ammonuium persulfate (APS)Sigma-AldrichA3678-100G
ATP, [α-32P] – 3000 Ci/mmolPerkin ElmerNEG003H250UC
Boric acid, granularGlentham Life SciencesGE4425
Bovine Serum Albumin (BSA)Roche10735094001
Bromophenol blueSigma-AldrichB0126-25G
Coplin jar
Dithiothreitol (DTT)Sigma-AldrichD0632-25G
DNA microarrayAgilent4x180K (AMADID #78366)
https://www.agilent.com
Ethylenediaminetetraacetic acid (EDTA)Acros OrganicsAC118430010
Fujifilm FLA-5100 phosphorimagerFUJIFILM Life Science
Glass slide staining rackThermo Scientific12869995If several slides are used
Lab rotatorThermo Scientific88880025
Magnesium chlorideSigma-Aldrich63064-500G
Microarray Hybridization ChamberAgilentG2534Ahttps://www.agilent.com/cs/library/usermanuals/Public/G2534-90004_HybridizationChamber_User.pdf
Microarray scanner (GenePix 4400A)Molecular Devices
Phosphate Buffered Saline (PBS)Sigma-AldrichP4417-100TAB
Potassium glutamateAlfa AesarA172232
Ribonucleotide Solution Mix (rNTPs)New England BioLabsN0466S
Salmon testes DNASigma-AldrichD1626-1G
Skim milk powderSigma-Aldrich70166-500G
Staining dishThermo Scientific12657696
Tetramethylethylenediamine (TEMED)Bio-Rad1610800
Tris base (2-Amino-2-(hydroxymethyl)-1,3-propanediol)Sigma-Aldrich93362-500G
Triton X-100Sigma-AldrichX100-500ML
Tween-20Sigma-AldrichP9416-50ML
UreaSigma-AldrichU6504-1KG
Xylene cyanolAlfa AesarB21530

References

  1. Berger, M. F., Bulyk, M. L. Protein binding microarrays (PBMs) for rapid, high-throughput characterization of the sequence specificities of DNA binding proteins. Methods in Molecular Biology. 338, 245-260 (2006).
  2. Berger, M. F., Bulyk, M. L.

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DNA PrimaseProtein DNA BindingMicroarrayHigh throughputSequence RecognitionRNA Primer FormationTranscription FactorsDNA BindingEnzymatic ActivitySequence Determinants

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