DamID
Encyclopedia
DamID is a molecular biology
protocol used to map the binding sites of DNA
- and chromatin
-binding protein
s in eukaryote
s. DamID identifies binding sites by expressing the proposed DNA-binding protein as a fusion protein
with DNA methyltransferase
. Binding of the protein of interest to DNA localizes the methyltransferase in the region of the binding site. Adenosine methylation does not occur naturally in eukaryotes and therefore adenine methylation in any region can be concluded to have been caused by the fusion protein, implying the region is located near a binding site. DamID is an alternate method to ChIP-on-chip
.
or DNA repair
systems. In Escherichia coli, adenine methylation is catalyzed by the adenine methyltransferase Dam (DNA adenine methyltransferase), which catalyses adenine methylation exclusively in the palindromic sequence GATC. Ectopic expression of Dam in eukaryotic cells leads to methylation of adenine in GATC sequences without any other noticeable side effect.
Based on this, DamID consists in fusing Dam to a protein of interest (usually a protein that interacts with DNA such as transcription factor
s) or a chromatin component. The protein of interest thus targets Dam to its cognate in vivo binding site, resulting in the methylation of neighboring GATCs. The presence of m6A, coinciding with the binding sites of the proteins of interest, is revealed by methyl PCR.
prior PCR amplification. This enzyme cuts non-methylated GATCs, ensuring that only fragments flanked by consecutive methylated GATCs are amplified.
against the protein of interest. On the one hand, this allows to map proteins for which no such antibody is available. On the other hand, this makes it impossible to specifically map posttranslationally modified
proteins.
Another fundamental difference is that ChIP assays where the protein of interests is at a given time, whereas DamID assays where it has been. The reason is that m6A stays in the DNA after the Dam fusion protein goes away. For proteins that are either bound or unbound on their target sites this does not change the big picture. However, this can lead to strong differences in the case of proteins that slide along the DNA (e.g. RNA polymerase).
experiments, the DNA of those plasmids is recovered along with the DNA of the transfected cells, meaning that fragments of the plasmid are amplified in the mePCR. Every sequence of the genome that shares homology or identity with the plasmid may thus appear to be bound by the protein of interest. In particular, this is true of the open reading frame
of the protein of interest, which is present in both the plasmid and the genome. In microarray
experiments, this bias can be used to ensure that the proper material was hybridized.
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
protocol used to map the binding sites of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
- and chromatin
Chromatin
Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
-binding protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s in eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s. DamID identifies binding sites by expressing the proposed DNA-binding protein as a fusion protein
Fusion protein
Fusion proteins or chimeric proteins are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins...
with DNA methyltransferase
DNA methyltransferase
In biochemistry, the DNA methyltransferase family of enzymescatalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions...
. Binding of the protein of interest to DNA localizes the methyltransferase in the region of the binding site. Adenosine methylation does not occur naturally in eukaryotes and therefore adenine methylation in any region can be concluded to have been caused by the fusion protein, implying the region is located near a binding site. DamID is an alternate method to ChIP-on-chip
ChIP-on-chip
ChIP-on-chip is a technique that combines chromatin immunoprecipitation with microarray technology . Like regular ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA in vivo...
.
Principle
N6-methyladenine (m6A) is the product of the addition of a methyl group (CH3) at position 6 of the adenine. This modified nucleotide is absent from the vast majority of eukaryotes, but is widespread in bacterial genomes, as part of the restriction modificationRestriction modification system
The restriction modification system is used by bacteria, and perhaps other prokaryotic organisms to protect themselves from foreign DNA, such as the one borne by bacteriophages. This phenomenon was first noticed in the 1950s. Certain bacteria strains were found to inhibit the growth of viruses...
or DNA repair
DNA repair
DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1...
systems. In Escherichia coli, adenine methylation is catalyzed by the adenine methyltransferase Dam (DNA adenine methyltransferase), which catalyses adenine methylation exclusively in the palindromic sequence GATC. Ectopic expression of Dam in eukaryotic cells leads to methylation of adenine in GATC sequences without any other noticeable side effect.
Based on this, DamID consists in fusing Dam to a protein of interest (usually a protein that interacts with DNA such as transcription factor
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
s) or a chromatin component. The protein of interest thus targets Dam to its cognate in vivo binding site, resulting in the methylation of neighboring GATCs. The presence of m6A, coinciding with the binding sites of the proteins of interest, is revealed by methyl PCR.
Methyl PCR (mePCR)
In this assay the genome is digested by DpnI, which cuts only methylated GATCs. Double-stranded adapters with a known sequence are then ligated to the ends generated by DpnI. A PCR with primers matching the adaptors is then carried out, leading to the specific amplification of genomic fragments flanked by methylated GATCs. In practice, ligation products are digested by DpnIIDpnII restriction endonuclease family
In molecular biology, the DpnII restriction endonuclease family is a family of restriction endonucleases which includes DpnII from Diplococcus pneumoniae. These enzymes recognise the double-stranded DNA unmethylated sequence GATC and cleave before G-1, where it encompasess the full length of the...
prior PCR amplification. This enzyme cuts non-methylated GATCs, ensuring that only fragments flanked by consecutive methylated GATCs are amplified.
Specificities of DamID versus ChIP (Chromatin Immuno-Precipitation)
ChIP is an alternative method to assay protein binding at specific loci of the genome. Unlike ChIP, DamID does not require a specific antibodyAntibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
against the protein of interest. On the one hand, this allows to map proteins for which no such antibody is available. On the other hand, this makes it impossible to specifically map posttranslationally modified
Posttranslational modification
Posttranslational modification is the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis, and thus gene expression, for many proteins....
proteins.
Another fundamental difference is that ChIP assays where the protein of interests is at a given time, whereas DamID assays where it has been. The reason is that m6A stays in the DNA after the Dam fusion protein goes away. For proteins that are either bound or unbound on their target sites this does not change the big picture. However, this can lead to strong differences in the case of proteins that slide along the DNA (e.g. RNA polymerase).
Plasmid methylation bias
Depending on how the experiment is carried out, DamID can be subject to plasmid methylation biases. Because plasmids are usually amplified in E. coli where Dam is naturally expressed, they are methylated on every GATC. In transient transfectionTransfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
experiments, the DNA of those plasmids is recovered along with the DNA of the transfected cells, meaning that fragments of the plasmid are amplified in the mePCR. Every sequence of the genome that shares homology or identity with the plasmid may thus appear to be bound by the protein of interest. In particular, this is true of the open reading frame
Open reading frame
In molecular genetics, an open reading frame is a DNA sequence that does not contain a stop codon in a given reading frame.Normally, inserts which interrupt the reading frame of a subsequent region after the start codon cause frameshift mutation of the sequence and dislocate the sequences for stop...
of the protein of interest, which is present in both the plasmid and the genome. In microarray
DNA microarray
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
experiments, this bias can be used to ensure that the proper material was hybridized.