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This lecture deals with
- Enzymes associated with DNA
- Sub-units of the enzymes and their functions
- Role in bacterial genome functioning
DNA associated enzymes
DNA associated enzymes
There are several enzymes, help to replicate, to modify the structure and to express of DNA in both prokaryotes and eukaryotes. The important classes of enzymes are detailed in this chapter.
DNA polymerase
DNA polymerase
It is an enzyme that helps catalyze the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases read an intact DNA strand as a template and uses it to synthesize the new strand. This process copies a piece of DNA. The newly polymerized molecule is complementary to the template strand and identical to the template's original partner strand. DNA polymerases use magnesium ions as cofactors.
- DNA polymerase can add free nucleotides to only the 3' end of the newly forming strand. This results in elongation of the new strand in a 5'-3' direction.
- No known DNA polymerase is able to begin a new chain.
- DNA polymerase can add a nucleotide onto only a preexisting 3'-OH group, and, therefore, needs a primer at which it can add the first nucleotide. Primers consist of RNA and/or DNA bases.
There are three types of DNA polymerases exist in the living systems.
DNA Polymerase I (or Pol I) is exclusively found in prokaryotes. It is composed of 928 amino acids, Discovered by Arthur Kornberg in 1956 and it was the first known DNA polymerase. It was initially characterized in E. coli, although it is ubiquitous in prokaryotes. In E.coli and many other bacteria, the gene that encodes Pol I is known as polA.
There are three major functions of PolI
- 5' to 3' (forward) DNA-Dependent DNA polymerase activity, requiring a primer site and a template strand.
- 3' to 5' (reverse) exonuclease activity (known as Proof reading ability)
- 5' to 3' (forward) exonuclease activity (Known as nick translation)
Nuclease activity refers cleaving / cutting the nucleotides in DNA. Exonuclease will remove nucleotides at the terminus of DNA, while endonucleases cut the DNA in the middle of the sequence.
Proof reading
When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA and excises/removes the mismatched base. Following base excision, the polymerase can re-insert the correct base and replication can continue. This process is called proof reading.
Nick-translation
The PolI can remove the short fragment of DNA from 5’ to 3’ direction, through exonuclease activity, and again replace with new nucleotides. This process needs a nick (referred as a break in phosphodiester bonds of single strand), hence it is referred as nick translation. This property of PolI could be useful for repair of damaged DNA due to chemicals and UV rays.
DNA polymerase II (also known as Pol II)
It is a prokaryotic DNA polymerase involved in DNA repair. The enzyme is 90 kDa in size and is coded by the polB gene. DNA PolII can synthesize DNA new base pairs at an average rate of between 40 and 50 nucleotides/second. It also has the proof reading ability (3’ to 5’ exonuclease activity). Strains lacking the gene show no defect in growth or replication. PolII differs from PolI in that it lacks a 5' to 3' exonuclease activity (nick translation).
DNA polymerase III (holoenzyme) is the primary enzyme complex involved in prokaryotic DNA replication.
Being the primary holoenzyme involved in replication activity, the DNA Pol III holoenzyme also has proofreading capabilities that correct replication mistakes by means of exonuclease activity working 3'to 5'. DNA Pol III is a component of the replisome, which is located at the replication fork.
How proof reading was done by PolI
Steps involved in nick translation by PolI
Replisome
Replisome
Replisome is a DNA-Protein (enzyme) complex formed during DNA replication.
The replisome is composed of the following:
Two DNA Pol III enzymes, each comprising α, ε and θ subunits. [The α subunit has the polymerase activity; the ε subunit has 3' to 5' exonuclease activity (Proof reading); the θ subunit stimulates the ε subunit's proofreading].
Twoβ units which act as sliding DNA clamps, they keep the polymerase bound to the DNA.
Two τ units which acts to dimerize two of the core enzymes (α, ε, and θ subunits).
One γ unit which acts as a clamp loader for the lagging strand Okazaki fragments, helping the two β subunits to form a unit and bind to DNA. The γ unit is made up of 5 γ subunits which include 3 γ subunits, 1 δ subunit, and 1 δ' subunit. The δ is involved in copying of the lagging strand.
Χ and Ψ which form a 1:1 complex and bind to γ or τ.
DNA polymerase III synthesizes base pairs at a rate of around 1000 nucleotides per second.DNA Pol III activity begins after strand separation at the origin of replication and PolIII needs a RNA primer to initiate the polymerization at 3’-OH position.
Sub-units of DNA Polymerse III (Key enzyme responsible for DNA replication)
Video describing the DNA polymerases in detail (Youtube)
Helicases (DnaB protein)
Helicases (DnaB protein)
Helicases are a class of enzymes separating two annealed nucleic acid strands using energy derived from ATP hydrolysis.Many cellular processes (DNA replication, transcription, translation, recombination, DNA repair, ribosome biogenesis) involve the separation of nucleic acid strands. Helicases are often utilized to separate strands of a DNA double helix using the energy from ATP hydrolysis, a process characterized by the breaking of hydrogen bonds between annealed nucleotide bases.
Helicase animation (Youtube)
DNA primase (DnaG protein)
DNA primase (DnaG protein)
Primase is an enzyme involved in the replication of DNA.Primase catalyzes the synthesis of a short RNA (or DNA in some organisms) segment called a primer complementary to assDNA template. Primase is of key importance in DNA replication because no known DNA polymerases can initiate the synthesis of a DNA strand without an initial RNA or DNA primer (for temporary DNA elongation).Primases in E. coli, synthesize around 2000 to 3000 primers at the rate of one primer per second.
In bacteria, primase binds to the DNA helicase forming a complex called the primosome.
About Primase (Youtube video)
Topoisomerases
Topoisomerases
The enzymes regulate the winding and unwinding of DNA are referred as topoisomerase. During DNA replication and transcription, DNA should be unwounded (otherwise relaxed) ahead of replisome or RNA polymerase respectively. For this, topoisomease(Type I) cuts one strand of a DNA double helix, relaxation occurs, and then the cut strand is reannealed. Cutting one strand allows the part of the molecule on one side of the cut to rotate around the uncut strand, thereby reducing stress from too much or too little twist in the helix. Such stress is introduced when the DNA strand is "supercoiled" or uncoiled to or from higher orders of coiling.
In contrast to this, topoisomerase (Type II) (also known as Gyrase) that introduces negative supercoils into DNA. It cuts both strands of one DNA double helix, passes another unbroken DNA helix through it, and then reanneals the cut strand. This process occurs in bacteria, whose single circular DNA is cut by DNA gyrase and the two ends are then twisted around each other to form supercoils. During DNA replication, they can also unwind the supercoiled DNA.
How topoisomerase supercoils DNA - Animation (Youtube)
DNA ligase
DNA ligase
It is an enzymethat joins single-stranded discontinuities in double stranded DNA molecules. DNA ligase has to create the final phosphodiester bond to complete the DNA synthesis. The enzyme is to form two covalent phosphodiester bonds between 3’ hydroxyl end of one nucleotide with 5’ phosphate end of another nucleotide. ATP is required for the ligation reaction.
Restriction Endonuclease
Restriction Endonuclease
Restriction endonuclease is an enzyme that cuts DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses. Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction. To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially and are routinely used for DNA modification and manipulation in laboratories.
EcoRI digestion produces "sticky" ends
Whereas, SmaI restriction enzyme cleavage produces "blunt" ends
Videos about restriction enzymes (Youtube)
Watch type-I and type-II restriction enzymes mode of action (Youtub
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