DNA Microarray

How do DNA microarrays work?

Basic Explanation

DNA microarrays work on the principal of base-pairing . Base-pairing allows probes to hybridize to targets on the microarray.

At a basic level microarrays are implemented as follows: a cell's RNA is extracted. This RNA (targets) is then multiplied, labeled with fluorescence and hybridized to existing DNA (probes) on the microarray. After hybridization, the probes that were hybridized with targets are fluorescent and a computer scanner is able to detect this fluorescence. Those probes that are fluorescent correspond to the genes that were expressed in the cell.

DNA FINGERPRINTING

What is DNA FINGERPRINTING?
 
The chemical structure of everyone's DNA is the same. The only difference between people (or any animal) is the order of the base pairs. There are so many millions of base pairs in each person's DNA that every person has a different sequence.
 

DNA Ligases

Ligation → a process that involves the formation of 4 phosphodiester (PD) bonds -- 2 at each end of the molecule between neighbouring 3'-OH and 5'-PO4

DNA Ligases

• Exquisite specificity for nicked DNA
• In E. coli:
  • homologous and monomeric
  • polypeptide chain with molecular weight of 75 KDa
  • require NAD+ as a co-factor
• In T4 phages, mammalian and plant cells:
  • polypeptide chain with molecular weight of 68 KDa
  • requires ATP as a co-factor and energy source

Reaction

1. Formation of an adenylate-enzyme complex involving NAD+/ATP
2. Release of nicotinaminde mononucleotide (NMN, in case of NAD+) or Pyrophosphate (Pi, in case of ATP) while an AMP residue binds covalently to the Ɛ-amino group of lysine in the enzyme via a phosphoric acid amide bond or "phosphoamidite bond". This occurs simultaneously with (1)
3. 5'-PO4 of DNA subsequently activated by transfer of adenylate residue
4. Nucleophilic substitution of 3'-OH at the activated 5'-PO4 residue
5. Release of AMP

Substrates

• Physiological → breakage point at a PD bond between neighboring 3'-OH and 5'-PO4 ends held together by an intact complementary strand (E. coli, T4)
• Open and staggered PD bonds formed through reassociation of the protruding termini of different DNA molecules held together by basepairing between 2, 3, 4 protruding nucleotides (again E. coli, T4)
• T4 DNA ligase → blunt end ligation of double stranded DNA (dsDNA) molecules
• T4 DNA ligase → nicks in the RNA chains of dsRNA-DNA hybrids (i.e annealing of RNA termini with DNA strands)

** single stranded (ss) polynucleotides are ligated by RNA ligase (eg. in T4)

Architecture

• 2 or more domains uniquely arranged
• 5 classes of motifs recently detected:
  1. NBD → nucleotide binding domain / AD → adenylation domain
  2. OB fold → oligomer binding fold
  3. ZFM → zinc finger motif
  4. HhH → helix-hairpin-helix motif
  5. BRCT → BRCA1 C-terminus domain

Adenylation domain

• Main enzyme has 2 domains:
  • Larger N-terminal → domain 1
  • In T7 (ATP dependent ligase):
    • 3 main anti-parallel β-sheets flanked by 6 α-helices
    • contains ATP-binding site situated in a pocket beneath the β-sheet
    • has intrinsic adenylation activity
  • In other NAD+ dependent ligases:
    • contains a sub-domain that is mainly α-helical
  • Smaller C-terminal → domain 2

OB fold domain

• Derivative of the Greek key motif
• Conserved, connects to Domain 1
• In T7:
  • binds dsDNA
  • dramatically enhances adenylation activity of Domain 1 by undergoing conformational change → 13A movement of Domain 2 towards Domain 1

Zinc Finger Motif

• 4 cystine conserved in C-terminal region of NAD+ dependent ligases which tetrahedrally ligand a zinc ion
• acts as a DNA recognition module that recognizes specific DNA sequences
• also plays a structural role in proteins by offering support for subdomain 3b and domain 4

HhH motif

• 4 copies of conserved HhH observed in NAD+ dependent ligases
• consists of 2 helices + 2 type II β-turns
• implicated in non-sequence specific binding
• in Tfi, 4 HhH form a compact structure (subdomain 2b)
• hairpins located in a linear chain at the bottom of a subdomain
• rich in positively charged residues
• forms 1 of 2 DNA binding sequences (eg. In Tif)

BCRT domain

• found in both NAD+ as well as ATP dependent ligases
• in Tfi, it consists of a 4-stranded parallel β-sheets flanked by 3 α-helices
• may act as a signal tranducer that transmits signals from DNA damage detectors to other components of the DNA repair machinery via specific protein-protein interactions
• mobile when in open conformation and restricted mobility in closed conformation
• acts as a gate which regulates DNA binding and release

Nick Recognition

• In T7 ligase:
  • dsDNA binds predominantly in the positively charged interdomain cleft lined by conserved motifs and residues with strong positive potential
  • domain 2 acts as a movable thumb that can open/close in response to ligand association/dissociation
  • domain 1 has a higher affinity for DNA than domain 2
  • however, both domains 1 + 2 are the minimal unit required for all the ATP dependent ligases and for NAD+ dependent bacterial DNA ligases
• Motif I → contains active site Lysine (K34, K116) which forms the covalent AMP adduct
• Motif III → contains a glutamate residue (E93, E114) which forms H-bonds with ribose of ATP
• Motif IIIa → contains Tyrosine (Y149, Y221) which is stacked against the adenine ring and the essential Lysine in motif V
• Motif V → contains the essential Lysine
• Larger ligases have additional domains* that enhance certain properties of the enzyme:
  • DNA binding
  • nick recognition
  • targeting of enzyme to sites of DNA damage
  • replication and recombination

*These additional domains are not directly involved in catalysis!

DNA Methylation controls Foxp3 gene expression

A basic science article was discussed at immunology journal club. It discusses the role of T regs and its how they come from naive T cells and what other factors triggers them.

The vast experiments done elegantly in the study concluded the following:

1. TGF-B induces FoxP3 expression in T regs but its unstable in vitro

2. Azacytidine derivatives( demethylation agent) stabalizes foxp3 expression in induced Tregs.

3. The T reg specific demthylated region (TSDR) determines transcriptional activity

4. Azacytidine promotes stable Foxp3 expression in vitro

5. In vivo induced Tregs exhibit stable Foxp3 expression and complete TSDR demethylation

6. Foxp3 Treg generated invivo by targeting of agonist ligands to dendritic cells showed long term survival in the absence of the inducing antigen and exhibited efficient TSDR demythylation.

Besides the specifics of epigenomic imprinting in the mehylation region of the T cell region which might be critical for T cell lineage, this paper I thought highlighted something very alarming. One is that TGF-B is an important promoter of T reg cells. There are drugs out there currently under development that are inhibitory to TGF-B for anti fibrotic effects. Again, we have to think  of these molecules as possible bimodal and perhaps they have different functions in different disease entities.

Second is that the dendritic cells might be important players in the long term survival of Tregs. Since there are studies that show that the patients that has increase Foxp3+ Tregs, those patients post transplant do better.

Perhaps, sustaining that positive effect should be the goal in transplantation. We can perhaps then achieve tolerance.

DNA Methylation in Bacteria

How do bacteria respond to environmental challenges and signals so quickly and effectively? In addition to genetic modifications there are a series of non genetic, or epigenetic, modifications. Genetic modifications change the sequence of nucleotides that, for instance, comprise a protein-coding gene. In that case the resulting protein is modified to better handle the environmental challenge. Epigenetics, on the other hand, involves various other types of modifications. For instance, the three-dimensional structures of proteins may be dramatically altered, or tiny chemical signals—methyl groups—may be added to certain proteins or DNA sequences. As with genetic modifications, many of the epigenetic modifications are heritable, so the adjustments are passed on to later generations.

Consider the methylation of DNA. This occurs at certain target sites along the DNA sequence where specific short DNA sequences appear. These sequences are found by special proteins as they move along the DNA. The special proteins search for these sequences and add a methyl group to the adenine base that appears within the sequence. The protein binds to the DNA, twists the helix so the adenine base rotates into a precisely shaped pocket in the protein, and the protein then facilitates the transfer of the methyl group from a short donor molecule to the adenine.

The short donor molecule does more, however, than just supply a methyl group. It also actually helps to control the special protein. How does this work? Of course the short donor molecule binds to the pocket of the special protein so the methyl group is ready for transfer. But the donor molecule also binds to another site on the protein. This binding serves to alter the structure of the protein, enhancing its function. So the special protein is designed to do its job when it is charged with a donor molecule.

But not all of the DNA target sequences are methylated. This complex DNA methylation function doesn’t occur if the target sequence is protected by another protein that binds to the sequence. This protein binds to some of these DNA target sequences but not all. The result is a particular DNA methylation pattern which influences which genes are expressed, and therefore how the bacteria interacts with the environment.

This DNA methylation pattern is propagated to the daughter cells when the bacteria divides. When such division occurs the DNA must, of course, be replicated. The double helix is separated and new complementary strands are synthesized on each strand. At the DNA target sequences there is an adenine on both strands. If both adenines are methylated, then after replication the two newly formed DNA helices will each contain only a single methylated adenine—the original adenines are methylated but the new adenines that were added are not.

These hemimethylated sequences are rectified by other proteins, which methylate the lone, unmethylated, adenines. The result is that after cell division, the two new bacteria cells have inherited the full DNA methylation pattern established in the original cell.

This is one small chapter of the epigenetics story that helps to explain the incredible adaptation capabilities we observe in both single and multiple cell organisms. The idea that such capabilities evolved is, of course, not motivated by science. Evolutionists once claimed that adaptation was obvious proof of evolution, but in fact biological adapation is yet another massive problem for evolution.

DNA Model Video

MineCraft DNA Model

3D DNA Model