DNA clamp
Encyclopedia
A 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. The clamp-polymerase protein–protein interactions are stronger and more specific than the direct interactions between the polymerase and the template DNA strand; because the rate-limiting step in the DNA synthesis reaction is the association of the polymerase with the DNA template, the presence of the sliding clamp dramatically increases the number of nucleotide
s that the polymerase can add to the growing strand per association event. The presence of the DNA clamp can increase the rate of DNA synthesis up to 1,000-fold compared with a nonprocessive polymerase.
s to the growing strand. The DNA clamp assembles on the DNA at the replication fork
and "slides" along the DNA with the advancing polymerase, aided by a layer of water
molecules in the central pore of the clamp between the DNA and the protein surface. Because of the toroidal
shape of the assembled multimer, the clamp cannot dissociate from the template strand without also dissociating into monomer
s.
The DNA clamp fold is found in bacteria
, archaea
, eukaryote
s and some viruses. In bacteria, the sliding clamp is a homodimer
composed of two identical beta subunits of DNA polymerase III
and hence is referred to as the beta clamp. In archaea and eukaryotes, it is a trimer composed of three molecules of PCNA. The T4 bacteriophage
also uses a sliding clamp, called gp45 that is a trimer similar in structure to PCNA but lacks sequence homology to either PCNA or the bacterial beta clamp.
The beta clamp is a specific DNA clamp and a subunit of the DNA polymerase III holoenzyme found in bacteria. Two beta subunits are assembled around the DNA by the gamma subunit and ATP hydrolysis; this assembly is called the pre-initiation complex
. After assembly around the DNA, the beta subunits' affinity for the gamma subunit is replaced by an affinity for the alpha and epsilon subunits, which together create the complete holoenzyme. DNA polymerase III is the primary enzyme complex involved in prokaryotic DNA replication
.
The gamma complex of DNA polymerase III, composed of γδδ'χψ subunits, catalyzes ATP
to chaperone two beta subunits to bind to DNA. Once bound to DNA, the beta subunits can freely slide along double stranded DNA. The beta subunits in turn bind the αε polymerase complex. The α subunit possesses DNA polymerase
activity and the ε subunit is a 3’-5’ exonuclease
.
The beta chain of bacterial DNA polymerase III is composed of three topologically non-equivalent domains
(N-terminal, central, and C-terminal). Two beta chain molecules are tightly associated to form a closed ring encircling duplex DNA.
called the proliferating cell nuclear antigen (PCNA
). The N-terminal and C-terminal domains of PCNA are topologically identical. Three PCNA molecules are tightly associated to form a closed ring encircling duplex DNA.
The sequence of PCNA is well conserved between plants and animals, indicating a strong selective pressure for structure conservation, and suggesting that this type of DNA replication mechanism is conserved throughout eukaryotes. Homologues of PCNA have also been identified in the archaea
(Euryarchaeota
and Crenarchaeota
) and in Paramecium bursaria Chlorella virus 1 (PBCV-1) and in nuclear polyhedrosis viruses
.
", which also disassemble the clamps after replication has completed. The binding sites for these initiator proteins overlap with the binding sites for the DNA polymerase, so the clamp cannot simultaneously associate with a clamp loader and with a polymerase. Thus the clamp will not be actively disassembled while the polymerase remains bound. Although DNA clamps play a less significant role in associating with other DNA-interacting proteins, such as nucleosome
assembly factors, Okazaki fragment
ligases, and DNA repair
proteins, all of these proteins also share a binding site on the DNA clamp that overlaps with the clamp loader site, ensuring that the clamp will not be removed while any enzyme is still working on the DNA. The activity of the clamp loader requires ATP hydrolysis
to "close" the clamp around the DNA.
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
fold
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...
that serves as a processivity
Processivity
In molecular biology, processivity is a measure of the average number of nucleotides added by a DNA polymerase enzyme per association/disassociation with the template. DNA polymerases associated with DNA replication tend to be highly processive, while those associated with DNA repair tend to have...
-promoting factor in 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...
. As a critical component of the DNA polymerase III holoenzyme
DNA polymerase III holoenzyme
DNA polymerase III holoenzyme is the primary enzyme complex involved in prokaryotic DNA replication. It was discovered by Thomas Kornberg and Malcolm Gefter in 1970. The complex has high processivity DNA polymerase III holoenzyme is the primary enzyme complex involved in prokaryotic DNA...
, the clamp protein binds DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
and prevents this enzyme
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...
from dissociating from the template 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...
strand. The clamp-polymerase protein–protein interactions are stronger and more specific than the direct interactions between the polymerase and the template DNA strand; because the rate-limiting step in the DNA synthesis reaction is the association of the polymerase with the DNA template, the presence of the sliding clamp dramatically increases 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 that the polymerase can add to the growing strand per association event. The presence of the DNA clamp can increase the rate of DNA synthesis up to 1,000-fold compared with a nonprocessive polymerase.
Structure
The DNA clamp fold is an α+β protein that assembles into a multimeric structure that completely encircles the DNA double helix as the polymerase adds nucleotideNucleotide
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 to the growing strand. The DNA clamp assembles on the DNA at the replication fork
Replication fork
The replication fork is a structure that forms within the nucleus during DNA replication. It is created by helicases, which break the hydrogen bonds holding the two DNA strands together. The resulting structure has two branching "prongs", each one made up of a single strand of DNA...
and "slides" along the DNA with the advancing polymerase, aided by a layer of water
Water
Water is a chemical substance with the chemical formula H2O. A water molecule contains one oxygen and two hydrogen atoms connected by covalent bonds. Water is a liquid at ambient conditions, but it often co-exists on Earth with its solid state, ice, and gaseous state . Water also exists in a...
molecules in the central pore of the clamp between the DNA and the protein surface. Because of the toroidal
Torus
In geometry, a torus is a surface of revolution generated by revolving a circle in three dimensional space about an axis coplanar with the circle...
shape of the assembled multimer, the clamp cannot dissociate from the template strand without also dissociating into monomer
Monomer
A monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer; the term "monomeric protein" may also be used to describe one of the proteins making up a multiprotein complex...
s.
The DNA clamp fold is found in bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
, archaea
Archaea
The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...
, eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s and some viruses. In bacteria, the sliding clamp is a homodimer
Protein dimer
In biochemistry, a dimer is a macromolecular complex formed by two, usually non-covalently bound, macromolecules like proteins or nucleic acids...
composed of two identical beta subunits of DNA polymerase III
DNA polymerase III holoenzyme
DNA polymerase III holoenzyme is the primary enzyme complex involved in prokaryotic DNA replication. It was discovered by Thomas Kornberg and Malcolm Gefter in 1970. The complex has high processivity DNA polymerase III holoenzyme is the primary enzyme complex involved in prokaryotic DNA...
and hence is referred to as the beta clamp. In archaea and eukaryotes, it is a trimer composed of three molecules of PCNA. The T4 bacteriophage
Enterobacteria phage T4
Enterobacteria phage T4 is a bacteriophage that infects E. coli bacteria. Its DNA is 169–170 kbp long, and is held in an icosahedral head. T4 is a relatively large phage, at approximately 90 nm wide and 200 nm long...
also uses a sliding clamp, called gp45 that is a trimer similar in structure to PCNA but lacks sequence homology to either PCNA or the bacterial beta clamp.
| Kingdom | Sliding clamp protein | Aggregation state | Associated polymerase |
|---|---|---|---|
| Bacteria Bacteria Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals... |
beta subunit of pol III | dimer | DNA polymerase III |
| Archaea Archaea The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon... |
archaeal PCNA PCNA Proliferating Cell Nuclear Antigen, commonly known as PCNA, is a protein that acts as a processivity factor for DNA polymerase δ in eukaryotic cells. It achieves this processivity by encircling the DNA, thus creating a topological link to the genome... |
trimer | pol ε |
| Eukaryote Eukaryote A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear... |
PCNA PCNA Proliferating Cell Nuclear Antigen, commonly known as PCNA, is a protein that acts as a processivity factor for DNA polymerase δ in eukaryotic cells. It achieves this processivity by encircling the DNA, thus creating a topological link to the genome... |
trimer | DNA polymerase delta DNA polymerase delta DNA polymerase delta is an enzyme complex found in eukaryotes that is involved in DNA replication and repair, and it consists of the proliferating cell nuclear antigen , the multisubunit replication factor C, and the 4 subunit polymerase complex: POLD1, POLD2, POLD3, and POLD4.... |
| Virus Virus A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea... |
gp43 / gp45 | timer | RB69 Pol / T4 Pol |
Bacterial
The beta clamp is a specific DNA clamp and a subunit of the DNA polymerase III holoenzyme found in bacteria. Two beta subunits are assembled around the DNA by the gamma subunit and ATP hydrolysis; this assembly is called the pre-initiation complex
Pre-replication complex
A pre-replication complex is a protein complex that forms at the origin of replication during the initiation step of DNA replication. Formation of the pre-RC is required for DNA replication to occur. Complete and faithful replication of the genome ensures that each daughter cell will carry the...
. After assembly around the DNA, the beta subunits' affinity for the gamma subunit is replaced by an affinity for the alpha and epsilon subunits, which together create the complete holoenzyme. DNA polymerase III is the primary enzyme 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...
.
The gamma complex of DNA polymerase III, composed of γδδ'χψ subunits, catalyzes ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
to chaperone two beta subunits to bind to DNA. Once bound to DNA, the beta subunits can freely slide along double stranded DNA. The beta subunits in turn bind the αε polymerase complex. The α subunit possesses DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
activity and the ε subunit is a 3’-5’ 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 ...
.
The beta chain of bacterial DNA polymerase III is composed of three topologically non-equivalent domains
Protein domain
A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural...
(N-terminal, central, and C-terminal). Two beta chain molecules are tightly associated to form a closed ring encircling duplex DNA.
Eukaryote
The sliding clamp in eukaryotes is assembled from a specific subunit of DNA polymerase deltaDNA polymerase delta
DNA polymerase delta is an enzyme complex found in eukaryotes that is involved in DNA replication and repair, and it consists of the proliferating cell nuclear antigen , the multisubunit replication factor C, and the 4 subunit polymerase complex: POLD1, POLD2, POLD3, and POLD4....
called the proliferating cell nuclear antigen (PCNA
PCNA
Proliferating Cell Nuclear Antigen, commonly known as PCNA, is a protein that acts as a processivity factor for DNA polymerase δ in eukaryotic cells. It achieves this processivity by encircling the DNA, thus creating a topological link to the genome...
). The N-terminal and C-terminal domains of PCNA are topologically identical. Three PCNA molecules are tightly associated to form a closed ring encircling duplex DNA.
The sequence of PCNA is well conserved between plants and animals, indicating a strong selective pressure for structure conservation, and suggesting that this type of DNA replication mechanism is conserved throughout eukaryotes. Homologues of PCNA have also been identified in the archaea
Archaea
The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...
(Euryarchaeota
Euryarchaeota
In the taxonomy of microorganisms, the Euryarchaeota are a phylum of the Archaea.The Euryarchaeota include the methanogens, which produce methane and are often found in intestines, the halobacteria, which survive extreme concentrations of salt, and some extremely thermophilic aerobes and anaerobes...
and Crenarchaeota
Crenarchaeota
In taxonomy, the Crenarchaeota has been classified as either a phylum of the Archaea kingdom or a kingdom of its own...
) and in Paramecium bursaria Chlorella virus 1 (PBCV-1) and in nuclear polyhedrosis viruses
Nuclear Polyhedrosis Virus
The nuclear polyhedrosis virus which belongs to the sub group Baculoviruses is a virus affecting insects, predominantly moths and butterflies. It has been used as a pesticide for crops infested by insects susceptible to contraction...
.
Viral
The viral gp45 sliding clamp subunit protein contains two domains. Each domain consists of two alpha helices and two beta sheets – the fold is duplicated and has internal pseudo two-fold symmetry. Three gp45 molecules are tightly associated to form a closed ring encircling duplex DNA.Assembly
Sliding clamps are loaded onto their associated DNA template strands by specialized proteins known as "sliding clamp loadersReplication factor C
The replication factor C, or RFC, is a five-subunit protein complex that is required for DNA replication.The subunits of this heteropentamer are named Rfc1, Rfc2, Rfc3, Rfc4, and Rfc5 . RFC is used in eukaryotic replication as a clamp loader, similar to the γ Complex in E. coli. Its role as clamp...
", which also disassemble the clamps after replication has completed. The binding sites for these initiator proteins overlap with the binding sites for the DNA polymerase, so the clamp cannot simultaneously associate with a clamp loader and with a polymerase. Thus the clamp will not be actively disassembled while the polymerase remains bound. Although DNA clamps play a less significant role in associating with other DNA-interacting proteins, such as nucleosome
Nucleosome
Nucleosomes are the basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound around a histone protein core. This structure is often compared to thread wrapped around a spool....
assembly factors, Okazaki fragment
Okazaki 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....
ligases, and DNA repair
DNA repair
DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1...
proteins, all of these proteins also share a binding site on the DNA clamp that overlaps with the clamp loader site, ensuring that the clamp will not be removed while any enzyme is still working on the DNA. The activity of the clamp loader requires ATP hydrolysis
ATP hydrolysis
ATP hydrolysis is the reaction by which chemical energy that has been stored and transported in the high-energy phosphoanhydridic bonds in ATP is released, for example in the muscles, to produce work. The product is ADP and an inorganic phosphate, orthophosphate...
to "close" the clamp around the DNA.