Related Experiment Video
Updated: Aug 11, 2026
09:16
Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Hemi-methylated oriC DNA binding activity found in non-specific acid phosphatase
E Reshetnyak1, E d'Alençon, R Kern
1Institut Jacques Monod, CNRS/Université Paris 6-7, France.
Molecular Microbiology
|February 13, 1999
Summary
Researchers identified a non-specific acid phosphatase (NAP) that binds to hemi-methylated DNA, crucial for Escherichia coli DNA replication initiation. This protein, encoded by aphA, shows preferential binding to hemi-methylated oriC DNA.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- DNA replication initiation in Escherichia coli is a complex process regulated by specific protein-DNA interactions.
- The origin of replication (oriC) plays a critical role in initiating DNA replication.
- DNA methylation is a key epigenetic mechanism influencing gene expression and DNA replication.
Purpose of the Study:
- To identify and characterize proteins involved in binding to hemi-methylated oriC DNA in Escherichia coli.
- To investigate the function of a novel protein identified through a lacZ-hobH fusion screen.
Main Methods:
- Purification of a protein exhibiting hemi-methylated DNA binding activity.
- DNA footprinting assays to assess protein-DNA interactions.
- Gel retardation assays to evaluate binding affinity to different DNA methylation states.
- Indirect immunofluorescence microscopy to determine protein localization within bacterial cells.
Main Results:
- A protein corresponding to non-specific acid phosphatase (NAP), encoded by aphA, was purified.
- Partially purified NAP protected hemi-methylated oriC DNA in footprinting assays.
- Purified NAP demonstrated specific binding to oriC DNA, with higher affinity for hemi-methylated forms in gel retardation assays.
- NAP was localized to discrete foci within E. coli cells, with distinct localization patterns depending on nucleoid number.
Conclusions:
- Non-specific acid phosphatase (NAP) is a DNA-binding protein that preferentially interacts with hemi-methylated oriC DNA.
- NAP may play a role in the initiation of DNA replication or other DNA-related processes in E. coli.
- The localization of NAP within the cell suggests a dynamic role in cell division and DNA segregation.
More Related Videos
08:07Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry
Published on: July 26, 2019
10:31A Liposome Membrane Permeability Assay for Investigating the Effects of Phosphatidylinositol Phosphate Groups on Membranotropic Action of Venom PLA2
Published on: September 26, 2025
Related Concept Videos
01:02Phosphorylation
The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
02:54Protein Kinases and Phosphatases
Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
02:04Covalently Linked Protein Regulators
Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.
These groups modify specific amino acids in a protein.
02:32Histone Modification
The histone proteins have a flexible N-terminal tail extending out from the nucleosome. These histone tails are often subjected to post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitination. Particular combinations of these modifications form “histone codes” that influence the chromatin folding and tissue-specific gene expression.
Acetylation
The enzyme histone acetyltransferase adds acetyl group to the histones. Another enzyme, histone deacetylase,...
Acetylation
The enzyme histone acetyltransferase adds acetyl group to the histones. Another enzyme, histone deacetylase,...
01:58Cell Specific Gene Expression
Multicellular organisms contain a variety of structurally and functionally distinct cell types, but the DNA in all the cells originated from the same parent cells. The differences in the cells can be attributed to the differential gene expression. Liver cells, whose functions include detoxification of blood, production of bile to metabolize fats, and synthesis of proteins essential for metabolism, must express a specific set of genes to perform their functions. Gene expression also varies with...
02:38Heterochromatin
The extent of chromatin compaction can be studied by staining chromatin using specific DNA binding dyes. Under the microscope, the dense-compacted regions that take up more dye are called heterochromatin. Heterochromatin is further classified into two forms – constitutive heterochromatin and facultative heterochromatin.
Constitutive heterochromatin: It is a highly compact region of chromatin that is mostly concentrated in the centromere and telomere. Unlike euchromatin, the amino acid at 9th...
Constitutive heterochromatin: It is a highly compact region of chromatin that is mostly concentrated in the centromere and telomere. Unlike euchromatin, the amino acid at 9th...