A subscription to JoVE is required to view this content. Sign in or start your free trial.
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.
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.
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....
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.
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.......
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.......
This research was supported by the ISRAEL SCIENCE FOUNDATION (grant no. 1023/18).
....Name | Company | Catalog Number | Comments |
40% acrylamide-bisacrylamide (19:1) solution | Merck | 1006401000 | |
95% formamide | Sigma-Aldrich | F9037-100ML | |
Alexa 488-conjugated anti-his antibody | Qiagen | 35310 | |
Ammonuium persulfate (APS) | Sigma-Aldrich | A3678-100G | |
ATP, [α-32P] – 3000 Ci/mmol | Perkin Elmer | NEG003H250UC | |
Boric acid, granular | Glentham Life Sciences | GE4425 | |
Bovine Serum Albumin (BSA) | Roche | 10735094001 | |
Bromophenol blue | Sigma-Aldrich | B0126-25G | |
Coplin jar | |||
Dithiothreitol (DTT) | Sigma-Aldrich | D0632-25G | |
DNA microarray | Agilent | 4x180K (AMADID #78366) https://www.agilent.com | |
Ethylenediaminetetraacetic acid (EDTA) | Acros Organics | AC118430010 | |
Fujifilm FLA-5100 phosphorimager | FUJIFILM Life Science | ||
Glass slide staining rack | Thermo Scientific | 12869995 | If several slides are used |
Lab rotator | Thermo Scientific | 88880025 | |
Magnesium chloride | Sigma-Aldrich | 63064-500G | |
Microarray Hybridization Chamber | Agilent | G2534A | https://www.agilent.com/cs/library/usermanuals/Public/G2534-90004_HybridizationChamber_User.pdf |
Microarray scanner (GenePix 4400A) | Molecular Devices | ||
Phosphate Buffered Saline (PBS) | Sigma-Aldrich | P4417-100TAB | |
Potassium glutamate | Alfa Aesar | A172232 | |
Ribonucleotide Solution Mix (rNTPs) | New England BioLabs | N0466S | |
Salmon testes DNA | Sigma-Aldrich | D1626-1G | |
Skim milk powder | Sigma-Aldrich | 70166-500G | |
Staining dish | Thermo Scientific | 12657696 | |
Tetramethylethylenediamine (TEMED) | Bio-Rad | 1610800 | |
Tris base (2-Amino-2-(hydroxymethyl)-1,3-propanediol) | Sigma-Aldrich | 93362-500G | |
Triton X-100 | Sigma-Aldrich | X100-500ML | |
Tween-20 | Sigma-Aldrich | P9416-50ML | |
Urea | Sigma-Aldrich | U6504-1KG | |
Xylene cyanol | Alfa Aesar | B21530 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved