10 DNA denaturation and its implications

Prof. Sunil Kumar Khare

  • Objectives
  • Introduction
  • In vivo DNA denaturation
  • In vitro DNA denaturation

 

Introduction

 

DNA denaturation and renaturation is a key phenomenon for many biological functions such as in situ hybridization, microarray and membrane hybridization. DNA denaturation is the process of separating single strand of DNA from double helix. Extensive reports have explained the importance of in vivo denaturation occurs inside the cell, it starts with the help of proteins, ligands and some drugs. The in vitro procedures for DNA denaturation is mainly done by heating, dimethyl sulfoxide (DMSO) and sonication.

 

In vivo denaturation of DNA

 

Helicase

 

In vitro denaturation participates in many essential cellular processes such as repair, DNA transcription and replication with the help if helicase. Inspite of vigorous investigations, the full mechanism of DNA denaturation by helicase is still not properly understood. However, we can conclude that there are two distinct models on the basis of movement of helicase to understand unwinding of DNA i.e rolling and creeping.

  1. Rolling: In this kind of DNA unwinding helicase attached at two distinct site on the DNA to be unzipped. Followed by incorporation of NTP at the attachment site, these NTP will relatively change the affinity of the helicase to bind with the target DNA and help in rolling of enzyme over DNA. The pre requisite for this kind of mechanism is the presence of two DNA binding and two NTP binding sites on an oligomeric helicase. According to this model the monomer of helicase has ability to bind ssDNA and dsDNA separately. One monomer binds behind the fork in ssDNA and other binds a head of the fork dsDNA and few monomers binds to the ssDNA-dsDNA junction itself. Then helicase denatures the DNA duplex at the junction by using energy releages from NTP hydrolysis. During this process, the protein DNA complex formed by monomer present towards downward stream is shifted forward to the upstream monomer and this cycle is completed by rolling of the enzyme over the helicase DNA complex.
  2. Creeping: According to this mechanism a monomer of hexameric helicase initially bound to the fork. Then second monomer gain energy by ATP hydrolysis to interact with the dsDNA (aprox 10 -15 bp ahead of fork). This phenomenon allow the denaturing of the duplex DNA between the DNA-binding sites of monomers 1. During above step retation of DNA is needed to allow complete untwisting of dsDNA. After this process helicase rolls to regain its original structure keeping its monomer 2 intact with the fork
  3. Fig1: Mechanism of DNA unwinding using helicase

 

Ligand based unzipping

 

Few compounds have ability to unzip the DNA. They intercalate between the minor and major groove and attach non-covalently with the walls of DNA. With the help of intercalative rings these compounds interact with the adjacent base pairs either in parallel or perpendicular ways which unwind the DNA helix by an angle less than 36°(36° is the angle between two adjacent base pairs). The value of angular rotation depends on the type of intercalating agent such as DNA alkylating agents unwinds the DNA by bending and flipping. On the basis of kind of type intercalation we can categorize them in two groups : mono- or bis- intercalators.

  1. Mono intercalating Compounds. These compounds have ability to intercalate in dsDNA or ssDNA. Example of such compounds are as follows: Acridine orange (AO), Formaldehyde, Diethylpyrocarbonate (DEPC), Ellipticine and adriamycin. Acridine orange (AO): When AO binds with the dsDNA it gives green fluorescence and gave red fluorescence while interacting with ssDNA and subsequently reduce the Tm even by 5-10 °C. Similarly, Ellipticine and adriamycin induce unwinding of the DNA helix and also reduce the Tm by approximately 5°C.
  2. Bis-Intercalating Agents. Bis –intercalating agents such as bis-acridine A, also have ability to unzip DNA. They have two intercalating core which are linked by polyammonium bridge. The bridge (polyammonium) destabilize the equilibrium of dsDNA and shift it towards ssDNA. Moreover in presence of light these compounds can induce photocleavage.

Physical method of DNA denaturation

 

Physical denaturation of DNA can be done by heating, beads mill, indirect and direct sonication described below.

 

Heating

 

Two different methods i.e heating only and heating with cold shock can be used to denature the DNA. During the heating process the energy of heat is utilized to pull the two strand of DNA apart making it single stranded. The temperature needed to pull two strands apart is the melting temperature (Tm). The melting temperature of DNA depends on the length and the composition of DNA. DNA with higher GC content usually has higher Tm due to presence of triple bonds between GC. The quantitative analysis of Tm of any naturally occurring DNA can be calculated by following formulae :

 

Tm = 81.5 oC + 0.41 ( % GC ) + 16.6 log [Na+ ]

 

However for short DNA hybrids, the formula is:

 

Tm = 2 oC (A+T) + 4 oC (G + C)

 

When we raise the temperature the double stranded DNA tend to dissociate which increase the absorbance at 260 nm this is called hyperchromicity (Fig 2). The denaturation of DNA can be implemented to identify the single nucleotide polymorphism. This information can also be used to know the nature of interacted material. For example certain intercalators intercalate between the DNA, interact with base pairs through pi stacking resulting in DNA stabilization which increase the Tm of DNA. Similarly, increasing salt concentration reduces the negative repulsion arises between the phosphate of DNA which leads to a raise in its melting temperature. Contrarily, pH shows negative effect on the stability of DNA and hence melting temperature decreases.

 

Fig: 2 Hyperchromic shift

Indirect and direct sonication

 

Denaturation by sonication either direct or indirect separated DNA with same mechanism. The movement of shock waves increase the random movement of molecules present in the medium, this generates the energy transfer which is used to denature the DNA. In case of indirect sonication the sound wave reach to the DNA by a medium such as ionized water, while in direct sonication the probe immersed directly in the solution containing DNA. However, denaturation by sonication is the combinational effects produce by heating as well as sound waves. According to Davis and Phillips DNA did not denature if we do sonication at lower temperature such as 0 – 2°C.