DNA polymerase III holoenzyme
Encyclopedia
DNA polymerase III holoenzyme is the primary enzyme
complex involved in prokaryotic DNA replication
. It was discovered by Thomas Kornberg
(son of Arthur Kornberg
) and Malcolm Gefter in 1970. The complex has high processivity (i.e. the number of nucleotide
s added per binding event) and, specifically referring to the replication of the E.coli
genome
, works in conjunction with four other DNA polymerases (Pol I, Pol II, Pol IV, and Pol V). 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'->5'. DNA Pol III is a component of the replisome
, which is located at the replication fork.
(an RNA polymerase
):
("!" for RNA
, '"$" for DNA
, "*" for polymerase
)
-------->
* * * *
! ! ! ! _ _ _ _
_ _ _ _ | RNA
| <--ribose
(sugar)-phosphate backbone
G U A U | Pol
| <--RNA primer
* * * * |_ _ _ _| <--hydrogen bonding
C A T A G C A T C C <--template
ssDNA
(single-stranded DNA
)
_ _ _ _ _ _ _ _ _ _ <--deoxyribose
(sugar)-phosphate backbone
$ $ $ $ $ $ $ $ $ $
moves forward, DNA polymerase III arrives at the RNA primer and begins replicating the DNA, adding onto the 3'OH of the primer:
* * * *
! ! ! ! _ _ _ _
_ _ _ _ | DNA
| <--ribose
(sugar)-phosphate backbone
G U A U | Pol
| <--RNA primer
* * * * |_III_ _| <--hydrogen bonding
C A T A G C A T C C <--template
ssDNA
(single-stranded DNA
)
_ _ _ _ _ _ _ _ _ _ <--deoxyribose
(sugar)-phosphate backbone
$ $ $ $ $ $ $ $ $ $
) of the DNA. DNA polymerase III has a high processivity and therefore, synthesizes DNA very quickly. This high processivity is due in part to the β-clamps that "hold" onto the DNA strands.
----------->
* * * *
! ! ! ! $ $ $ $ $ $ _ _ _ _
_ _ _ _ _ _ _ _ _ _| DNA
| <--deoxyribose
(sugar)-phosphate backbone
G U A U C G T A G G| Pol
| <--RNA primer
* * * * * * * * * *|_III_ _| <--hydrogen bonding
C A T A G C A T C C <--template
ssDNA
(single-stranded DNA
)
_ _ _ _ _ _ _ _ _ _ <--deoxyribose
(sugar)-phosphate backbone
$ $ $ $ $ $ $ $ $ $
via the process of nick translation
. The removal of the RNA primer allows DNA ligase
to ligate the DNA-DNA nick between the new fragment and the previous strand. DNA polymerase I & III, along with many other enzymes are all required for the high fidelity, high-processivity of DNA replication.
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
complex involved in prokaryotic DNA replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
. It was discovered by Thomas Kornberg
Thomas B. Kornberg
Thomas Bill Kornberg is an American biochemist who was the first person to purify and characterise DNA polymerase II and DNA polymerase III. He is currently a Professor of biochemistry and biophysics at the University of California, San Francisco working on Drosophila melanogaster development.His...
(son of Arthur Kornberg
Arthur Kornberg
Arthur Kornberg was an American biochemist who won the Nobel Prize in Physiology or Medicine 1959 for his discovery of "the mechanisms in the biological synthesis of deoxyribonucleic acid " together with Dr. Severo Ochoa of New York University...
) and Malcolm Gefter in 1970. The complex has high processivity (i.e. the number of nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
s added per binding event) and, specifically referring to the replication of the E.coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
, works in conjunction with four other DNA polymerases (Pol I, Pol II, Pol IV, and Pol V). 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
Exonuclease
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle ...
activity working 3'->5'. DNA Pol III is a component of the replisome
Replisome
The replisome is a complex molecular machine that carries out replication of DNA. It is made up of a number of subcomponents that each provide a specific function during the process of replication.-Major components:...
, which is located at the replication fork.
Components
The replisome is composed of the following:- 2 DNA Pol III enzymes, each comprising α, ε and θ subunits.
- the α subunit has the polymerase activity.
- the ε subunit as 3'-5' exonuclease activity.
- the θ subunit stimulates the ε subunit's proofreading.
- 2 β units which act as sliding DNA clampDNA clampA DNA clamp, also known as a sliding clamp, is a protein fold that serves as a processivity-promoting factor in DNA replication. As a critical component of the DNA polymerase III holoenzyme, the clamp protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand...
s, they keep the polymerase bound to the DNA. - 2 τ units which acts to dimerize two of the core enzymes (α, ε, and θ subunits).
- 1 γ unit which acts as a clamp loader for the lagging strand Okazaki fragmentOkazaki fragmentOkazaki fragments are short molecules of single-stranded DNA that are formed on the lagging strand during DNA replication. They are between 1,000 to 2,000 nucleotides long in Escherichia coli and are between 100 to 200 nucleotides long in eukaryotes....
s, 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.
Activity
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. Because DNA synthesis cannot start de novo, an RNA primer, complementary to part of the single-stranded DNA, is synthesized by primasePrimase
DNA primase is an enzyme involved in the replication of DNA.Primase catalyzes the synthesis of a short RNA segment called a primer complementary to a ssDNA template...
(an RNA polymerase
RNA polymerase
RNA polymerase is an enzyme that produces RNA. In cells, RNAP is needed for constructing RNA chains from DNA genes as templates, a process called transcription. RNA polymerase enzymes are essential to life and are found in all organisms and many viruses...
):
("!" for RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
, '"$" for 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...
, "*" for polymerase
Polymerase
A polymerase is an enzyme whose central function is associated with polymers of nucleic acids such as RNA and DNA.The primary function of a polymerase is the polymerization of new DNA or RNA against an existing DNA or RNA template in the processes of replication and transcription...
)
-------->
* * * *
! ! ! ! _ _ _ _
_ _ _ _ | RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
| <--ribose
Ribose
Ribose is an organic compound with the formula C5H10O5; specifically, a monosaccharide with linear form H––4–H, which has all the hydroxyl groups on the same side in the Fischer projection....
(sugar)-phosphate backbone
G U A U | Pol
Polymerase
A polymerase is an enzyme whose central function is associated with polymers of nucleic acids such as RNA and DNA.The primary function of a polymerase is the polymerization of new DNA or RNA against an existing DNA or RNA template in the processes of replication and transcription...
| <--RNA primer
* * * * |_ _ _ _| <--hydrogen bonding
C A T A G C A T C C <--template
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...
ssDNA
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...
(single-stranded 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...
)
_ _ _ _ _ _ _ _ _ _ <--deoxyribose
Deoxyribose
Deoxyribose, more, precisely 2-deoxyribose, is a monosaccharide with idealized formula H---3-H. Its name indicates that it is a deoxy sugar, meaning that it is derived from the sugar ribose by loss of an oxygen atom...
(sugar)-phosphate backbone
$ $ $ $ $ $ $ $ $ $
Addition onto 3'OH
As replication progresses and the replisomeReplisome
The replisome is a complex molecular machine that carries out replication of DNA. It is made up of a number of subcomponents that each provide a specific function during the process of replication.-Major components:...
moves forward, DNA polymerase III arrives at the RNA primer and begins replicating the DNA, adding onto the 3'OH of the primer:
* * * *
! ! ! ! _ _ _ _
_ _ _ _ | 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...
| <--ribose
Ribose
Ribose is an organic compound with the formula C5H10O5; specifically, a monosaccharide with linear form H––4–H, which has all the hydroxyl groups on the same side in the Fischer projection....
(sugar)-phosphate backbone
G U A U | Pol
Polymerase
A polymerase is an enzyme whose central function is associated with polymers of nucleic acids such as RNA and DNA.The primary function of a polymerase is the polymerization of new DNA or RNA against an existing DNA or RNA template in the processes of replication and transcription...
| <--RNA primer
* * * * |_III_ _| <--hydrogen bonding
C A T A G C A T C C <--template
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...
ssDNA
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...
(single-stranded 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...
)
_ _ _ _ _ _ _ _ _ _ <--deoxyribose
Deoxyribose
Deoxyribose, more, precisely 2-deoxyribose, is a monosaccharide with idealized formula H---3-H. Its name indicates that it is a deoxy sugar, meaning that it is derived from the sugar ribose by loss of an oxygen atom...
(sugar)-phosphate backbone
$ $ $ $ $ $ $ $ $ $
Synthesis of DNA
DNA polymerase III will then synthesize a continuous or discontinuous strand of DNA, depending if this is occurring on the leading or lagging strand (Okazaki fragmentOkazaki fragment
Okazaki fragments are short molecules of single-stranded DNA that are formed on the lagging strand during DNA replication. They are between 1,000 to 2,000 nucleotides long in Escherichia coli and are between 100 to 200 nucleotides long in eukaryotes....
) of the DNA. DNA polymerase III has a high processivity and therefore, synthesizes DNA very quickly. This high processivity is due in part to the β-clamps that "hold" onto the DNA strands.
----------->
* * * *
! ! ! ! $ $ $ $ $ $ _ _ _ _
_ _ _ _ _ _ _ _ _ _| 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...
| <--deoxyribose
Deoxyribose
Deoxyribose, more, precisely 2-deoxyribose, is a monosaccharide with idealized formula H---3-H. Its name indicates that it is a deoxy sugar, meaning that it is derived from the sugar ribose by loss of an oxygen atom...
(sugar)-phosphate backbone
G U A U C G T A G G| Pol
Polymerase
A polymerase is an enzyme whose central function is associated with polymers of nucleic acids such as RNA and DNA.The primary function of a polymerase is the polymerization of new DNA or RNA against an existing DNA or RNA template in the processes of replication and transcription...
| <--RNA primer
* * * * * * * * * *|_III_ _| <--hydrogen bonding
C A T A G C A T C C <--template
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...
ssDNA
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...
(single-stranded 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...
)
_ _ _ _ _ _ _ _ _ _ <--deoxyribose
Deoxyribose
Deoxyribose, more, precisely 2-deoxyribose, is a monosaccharide with idealized formula H---3-H. Its name indicates that it is a deoxy sugar, meaning that it is derived from the sugar ribose by loss of an oxygen atom...
(sugar)-phosphate backbone
$ $ $ $ $ $ $ $ $ $
Removal of primer
After replication of the desired region, the RNA primer is removed by DNA polymerase IDNA polymerase I
DNA Polymerase I is an enzyme that participates in the process of DNA replication in prokaryotes. It is composed of 928 amino acids, and is an example of a processive enzyme - it can sequentially catalyze multiple polymerisations. Discovered by Arthur Kornberg in 1956, it was the first known...
via the process of nick translation
Nick translation
Nick translation was developed in 1977 by Rigby and Paul Berg. It is a tagging technique in molecular biology in which DNA Polymerase I is used to replace some of the nucleotides of a DNA sequence with their labeled analogues, creating a tagged DNA sequence which can be used as a probe in...
. The removal of the RNA primer allows DNA ligase
DNA ligase
In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...
to ligate the DNA-DNA nick between the new fragment and the previous strand. DNA polymerase I & III, along with many other enzymes are all required for the high fidelity, high-processivity of DNA replication.
External links
- Overview at Oregon State UniversityOregon State UniversityOregon State University is a coeducational, public research university located in Corvallis, Oregon, United States. The university offers undergraduate, graduate and doctoral degrees and a multitude of research opportunities. There are more than 200 academic degree programs offered through the...