Interaction of the double helix with small molecules and ions, Replication of DNA.

Mitesh Shrestha

Interaction of the double helix with small molecules and ions

• The three dimensional structure of nucleic acids, including the iconic DNA double helix, are the result of both their primary chemical structure and the interactions of these polymers with counter ions and solvent molecules.

• The negative charges that exist at each phosphate group along a nucleic acid backbone requires that cations in solution (e.g., sodium and magnesium ions) shield \ electrostatic repulsions that would otherwise prohibit the close approach of phosphate groups in secondary and higher order structures.

Interaction of the double helix with small molecules and ions

• DNA as carrier of genetic information is a major target for drug interaction because of the ability to interfere with transcription (gene expression and protein synthesis) and DNA replication, a major step in cell growth and division.

• There are three principally different ways of drug binding.

– First, through control of transcription factors and polymerases. Here, the drugs interact with the proteins that bind to DNA.

– Second, through RNA binding to DNA double helices to form nucleic acid triple helical structures or RNA hybridization (sequence specific binding) to exposed DNA single strand regions forming DNA/RNA hybrids that may interfere with transcriptional activity.

– Third, small aromatic ligand molecules that bind to DNA double helical structures by

• (i) intercalating between stacked base pairs thereby distorting the DNA backbone conformation and interfering with DNA protein interaction or

• (ii) the minor groove binders. This cause little distortion of the DNA backbone. Both work through non covalent interaction.

Modeling DNA ligand interaction of intercalating ligands

• The following properties have been identified as important for the successful modeling of ligand DNA interaction:

– degrees of freedom

– role of base pair sequence

– counter ion effects

– role of solvent ligand - receptor binding

Degrees of freedom

Modeling DNA ligand interaction of minor groove binders

• Hairpin minor grove binding molecules have been identified and synthesized that bind to GC rich nucleotide sequences.

• Hairpin polyamides are linked systems that exploit a set of simple recognition rules for DNA base pairs through specific orientation of imidazole (Im) and pyrrole (Py) rings.

• The hairpin polyamides originated from the discovery of the threering Im-PyPy molecule that bound to minor groove DNA as an antiparallel side by side dimer.

Drugs that form covalent bonds with DNA targets

• Drugs that interfere with DNA function by chemically modifying specific nucleotides are Mitomycin C, Cisplatin, and Anthramycin.

– Mitomycin C is a well characterized antitumor antibiotic which forms a

covalent interaction with DNA after reductive activation. The activated antibiotic forms a crosslinking structure between guanine bases on adjacent strands of DNA thereby inhibiting single strand formation (this is essential for mRNA transcription and DNA replication).

– Anthramycin is an antitumor antibiotic which bind covalently to N2 of guanine located in the minor groove of DNA. Anthramycin has a preference of purineG-purine sequences (purines are adenine and guanine) with bonding to the middle G.

– Cisplatin is a transition metal complex cisdiaminedichloroplatinum and clinically used as anticancer drug. The effect of the drug is due to the ability to platinate the N7 of guanine on the major groove site of DNA double helix. This chemical modification of platinum atom crosslinks two adjacent guanines on the same DNA strand interfering with the mobility of DNA polymerases

Intercalating Agents

• A group of aromatic organic molecules

• Roughly the same dimensions as nitrogenous base pair

• Intercalate or wedge between the base pair

• Insertion stretches the DNA duplex and the DNA polymerase is fooled into inserting an extra base opposite an intercalating molecule.

• Examples: – 2,8-Diamino acridine (proflavin) – Acridine orange – Ethidium Bromide

Intercalating Agents

Interactions of metal ions with DNA

Interactions of metal ions with DNA

• Metal ions have different affinity for different binding sites and so they are classified into two groups, soft and hard ions.

• Soft ions are more polarizable in comparison to the hard ions and therefore the interactions between soft ions and soft ligands have more covalent character. On contrary, interactions between hard ions and hard ligands are usually electrostatic.

• Hard ions: – Li+, Na+, K+, Mg2+, Ca2+, Mn2+, Ba2+, Zn2+ and Co3+.

• Soft ions – Cu+, Ag+, Hg+, Hg2+, Cd2+ and Pt2+.

Interactions of metal ions with DNA

• The ability of cation to bind specifically to nucleic acids depends on its valence, hydration free energy and its coordination geometry.

• In aqueous solutions cations are surrounded by at least three hydration layers and can bind tightly to DNA as partially dehydrated or fully hydrated.

• Generally, there are three distinct types of interaction between cations and nucleic acids.

• In diffuse binding, fully hydrated cations interact with DNA via non-specific long-range electrostatic interactions.

• In the case of non-specific site binding, hydrated cations interact with nucleic acid structure (through hydrogen bonding of water molecules),

• In the case of specific binding at least one cation aqua ligand is replaced by a ligand from nucleic acid structure

Replication of DNA

• Biological process of producing two identical replicas of DNA from one original DNA molecule.

• Occurs in all living organisms and is the basis for biological inheritance.

• DNA is made up of a double helix of two complementary strands.

• During replication, these strands are separated.

• Each strand of the original DNA molecule then serves as a template for the production of its counterpart, a process referred to as semiconservative replication.

• Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.

Three main features of the DNA synthesis reaction: 1. DNA polymerase I catalyzes formation of phosphodiester bond between 3’-OH of the deoxyribose (on the last nucleotide) and the 5’-phosphate of the dNTP. • Energy for this reaction is derived from the release of two of the three phosphates of the

dNTP. 2. DNA polymerase “finds” the correct complementary dNTP at each step in the lengthening

process.

• rate ≤ 800 dNTPs/second • low error rate

3. Direction of synthesis is 5’ to 3’