Primosome
Encyclopedia
In molecular biology, a primosome is a protein
complex responsible for creating RNA
primers on single stranded DNA
during DNA replication
.
The primosome consists of seven proteins: DnaG
primase
, DnaB helicase
, DnaC
helicase assistant, DnaT, PriA
, Pri B, and PriC. The primosome is utilized once on the leading strand of DNA
and repeatedly, initiating each Okazaki fragment
, on the lagging DNA
strand. Initially the complex formed by PriA, PriB, and PriC binds to DNA. Then the DnaB-DnaC helicase complex attaches along with DnaT. This structure is referred to as the pre-primosome. Finally, DnaG
will bind to the pre-primosome forming a complete primosome. The primosome attaches 1-10 RNA nucleotides to the single stranded DNA creating a DNA-RNA hybrid. This sequence of RNA is used as a primer to initiate DNA polymerase III. The RNA bases are ultimately replaced with DNA bases by RNase H
nuclease (eukaryotes) or DNA polymerase I
nuclease (prokaryotes). DNA Ligase
then acts to join the two ends together.
Assembly of the Escherichia coli primosome requires six proteins, PriA, PriB, PriC, DnaB, DnaC, and DnaT,
acting at a primosome assembly site (pas) on an SSBcoated single-stranded (8s) DNA. Assembly is initiated
by interactions of PriA and PriB with ssDNA and the pas. PriC, DnaB, DnaC, and DnaT then act on the PriAPriB-
DNA complex to yield the primosome.
Primosomes are nucleoproteins assemblies that activate DNA replication forks. Their primary role is to recruit the replicative helicase onto single-stranded DNA. The "replication restart" primosome, defined in Escherichia coli, is involved in the reactivation of arrested replication forks. Binding of the PriA protein to forked DNA triggers its assembly. PriA is conserved in bacteria, but its primosomal partners are not. In Bacillus subtilis, genetic analysis has revealed three primosomal proteins, DnaB, DnaD, and DnaI, that have no obvious homologues in E. coli. Interestingly, they are involved in primosome function both at arrested replication forks and at the chromosomal origin. Our biochemical analysis of the DnaB and DnaD proteins unravels their role in primosome assembly. They are both multimeric and bind individually to DNA. Furthermore, DnaD stimulates DnaB binding activities. DnaD alone and the DnaD/DnaB pair interact specifically with PriA of B. subtilis on several DNA substrates. This suggests that the nucleoprotein assembly is sequential in the PriA, DnaD, DnaB order. The preferred DNA substrate mimics an arrested DNA replication fork with unreplicated lagging strand, structurally identical to a product of recombinational repair of a stalled replication fork.
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...
complex responsible for creating RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
primers on 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...
during 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 primosome consists of seven proteins: DnaG
DnaG
DnaG is a bacterial primase which synthesizes short RNA oligonucleotides during DNA replication. These RNA oligonucleotides serve as primers for DNA synthesis by bacterial DNA polymerase Pol III. On one of the two parental strands, called the lagging strand, the primase makes a primer every few...
primase
Primase
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...
, DnaB helicase
Helicase
Helicases are a class of enzymes vital to all living organisms. They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands using energy derived from ATP hydrolysis.-Function:Many cellular processes Helicases are a...
, DnaC
DnaC
dnaC is a bacterial gene that codes for protein DnaC. DnaC, also known as the helicase loader, is a regulator of DnaB. DnaC forms a complex with DnaB, thus enabling DnaB to perform its functions in bacterial DNA replication. This interaction of DnaC with DnaB requires the hydrolysis of ATP....
helicase assistant, DnaT, PriA
Pría
Pría is one of 28 parishes in Llanes, a municipality within the province and autonomous community of Asturias, in northern Spain.-Villages:*Pria de la Cabuerna*Garaña*La Pesa*Llames*Piñeres*Silviella*Villanueva...
, Pri B, and PriC. The primosome is utilized once on the leading strand of 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...
and repeatedly, initiating each 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....
, on the lagging 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. Initially the complex formed by PriA, PriB, and PriC binds to DNA. Then the DnaB-DnaC helicase complex attaches along with DnaT. This structure is referred to as the pre-primosome. Finally, DnaG
DnaG
DnaG is a bacterial primase which synthesizes short RNA oligonucleotides during DNA replication. These RNA oligonucleotides serve as primers for DNA synthesis by bacterial DNA polymerase Pol III. On one of the two parental strands, called the lagging strand, the primase makes a primer every few...
will bind to the pre-primosome forming a complete primosome. The primosome attaches 1-10 RNA nucleotides to the single stranded DNA creating a DNA-RNA hybrid. This sequence of RNA is used as a primer to initiate DNA polymerase III. The RNA bases are ultimately replaced with DNA bases by RNase H
RNase H
The enzyme RNase H is a non-specific endonuclease and catalyzes the cleavage of RNA via a hydrolytic mechanism. Members of the RNase H family can be found in nearly all organisms, from archaea to bacteria and eukaryota....
nuclease (eukaryotes) or DNA polymerase I
DNA 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...
nuclease (prokaryotes). 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...
then acts to join the two ends together.
Assembly of the Escherichia coli primosome requires six proteins, PriA, PriB, PriC, DnaB, DnaC, and DnaT,
acting at a primosome assembly site (pas) on an SSBcoated single-stranded (8s) DNA. Assembly is initiated
by interactions of PriA and PriB with ssDNA and the pas. PriC, DnaB, DnaC, and DnaT then act on the PriAPriB-
DNA complex to yield the primosome.
Primosomes are nucleoproteins assemblies that activate DNA replication forks. Their primary role is to recruit the replicative helicase onto single-stranded DNA. The "replication restart" primosome, defined in Escherichia coli, is involved in the reactivation of arrested replication forks. Binding of the PriA protein to forked DNA triggers its assembly. PriA is conserved in bacteria, but its primosomal partners are not. In Bacillus subtilis, genetic analysis has revealed three primosomal proteins, DnaB, DnaD, and DnaI, that have no obvious homologues in E. coli. Interestingly, they are involved in primosome function both at arrested replication forks and at the chromosomal origin. Our biochemical analysis of the DnaB and DnaD proteins unravels their role in primosome assembly. They are both multimeric and bind individually to DNA. Furthermore, DnaD stimulates DnaB binding activities. DnaD alone and the DnaD/DnaB pair interact specifically with PriA of B. subtilis on several DNA substrates. This suggests that the nucleoprotein assembly is sequential in the PriA, DnaD, DnaB order. The preferred DNA substrate mimics an arrested DNA replication fork with unreplicated lagging strand, structurally identical to a product of recombinational repair of a stalled replication fork.