Pre-replication complex
Encyclopedia
A pre-replication complex (pre-RC) 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 same genetic information as the parent cell. Accordingly, formation of the pre-RC is a very important part of the cell cycle
.
In prokaryotes, the main component of the pre-RC is DnaA
. The pre-RC is complete when DnaA occupies all of its binding sites within the prokaryotic origin of replication (oriC).
The archaeal pre-RC is very different from the prokaryotic pre-RC and can serve as a simplified model of the eukaryotic pre-RC. It is composed of a single origin recognition complex
(ORC) protein, Cdc6
, and a homohexamer of the Mini Chromosome Maintenance
(MCM) protein.
The eukaryotic pre-RC is the most complex and highly regulated pre-RC. In most eukaryotes it is composed of six ORC proteins (ORC1-6), Cdc6, Cdt1, and a heterohexamer of the six MCM proteins (MCM2-7). The MCM heterohexamer arguably arose via MCM gene duplication events and subsequent divergent evolution. The pre-RC of Schizosaccharomyces pombe
(S. pombe) is notably different from that of other eukaryotes; Cdc6 is replaced by the homologous Cdc18 protein. Sap1 is also included in the S. pombe pre-RC because it is required for Cdc18 binding. The pre-RC of Xenopus laevis (X. laevis) also has an additional protein, MCM9, which helps load the MCM heterohexamer onto the origin of replication.
In prokaryotes, origin recognition is accomplished by DnaA. DnaA binds tightly to a 9-base pair consensus sequence in oriC; 5' - TTATCCACA - 3'. There are 5 such 9-bp sequences (R1-R5) and 4 non-consensus sequences (I1-I4) within oriC that DnaA binds with differential affinity. DnaA binds R4, R1, and R2 with high affinity and R5, I1, I2, I3, and R3 with lesser affinity. The pre-RC is complete when DnaA occupies all of the high and low affinity 9-bp binding sites
Archaea have 1-3 origins of replication. The origins are generally AT-rich tracts that vary based on the archaeal species. The singular archaeal ORC protein recognizes the AT-rich tracts and binds DNA in an ATP-dependent fashion.
Eukaryotes typically have multiple origins of replication; at least one per chromosome. Saccharomyces cerevisiae
(S. cerevisiae) is the only known eukaryote with a defined initiation sequence TTTTTATG/ATTTA/T. This initiation sequence is recognized by ORC1-5. ORC6 is not known to bind DNA in S. cerevisiae. Initiation sequences in S. pombe and higher eukaryotes are not well defined. However, the initiation sequences are generally either AT-rich or exhibit bent or curved DNA topology. The ORC4 protein is known to bind the AT-rich portion of the origin of replication in S. pombe using AT hook motifs. The mechanism of origin recognition in higher eukaryotes is not well understood but it is thought that the ORC1-6 proteins depend on unusual DNA topology for binding.
of the cell cycle when cyclin-dependent kinase
(CDK) activity is low. This timing and other regulatory mechanisms ensure that DNA replication will only occur once per cell cycle. Assembly of the pre-RC relies on prior origin recognition, either by DnaA in prokaryotes or by ORC in archaea and eukaryotes.
The pre-RC of prokaryotes is complete when DnaA occupies all possible binding sites within the oriC.
The pre-RC of archaea requires ORC binding of the origin. After this, Cdc6 and the MCM homohexameric complex bind in a sequential fashion.
Eukaryotes have the most complex pre-RC. After ORC1-6 bind the origin of replication, Cdc6 is recruited. Cdc6 recruits the licensing factor Cdt1 and MCM2-7. Cdt1 binding and ATP hydrolysis by the ORC and Cdc6 load MCM2-7 onto DNA. There is a stoichiometric excess of the MCM proteins over the ORC and Cdc6 proteins, indicating that there may be multiple MCM heterohexamers bound to each origin of replication.
In prokaryotes, DnaA hydrolyzes ATP in order to unwind DNA at the oriC. This denatured region is accessible to the DnaB helicase
and DnaC
helicase loader. Single-strand binding protein
s stabilize the newly formed replication bubble and interact with the DnaG
primase
. DnaG recruits the replicative DNA polymerase
III, and replication begins.
In eukaryotes, MCM heterohexamer is phosphorylated by CDC7 and CDK, which displaces Cdc6 and recruits MCM10
. MCM10 cooperates with MCM2-7 in the recruitment of Cdc45. Cdc45 then recruits key components of the replisome
; the replicative DNA polymerase α and its primase. DNA replication can then begin.
, and dissociating ORC1-6 from chromatin
via phosphorylation
. Metazoans have a fourth mechanism to prevent re-replication; during S and G2 geminin
binds to Cdt1 and inhibits Cdt1 from loading MCM2-7 onto the origin of replication.
. Known mutations are in the ORC1
, ORC4, ORC6, CDT1, and CDC6 genes. The disease phenotype is probably due to reduced ability of cells to proliferate, reduced cell number, and general growth failure.
Protein complex
A multiprotein complex is a group of two or more associated polypeptide chains. If the different polypeptide chains contain different protein domain, the resulting multiprotein complex can have multiple catalytic functions...
that forms at the origin of replication
Origin of replication
The origin of replication is a particular sequence in a genome at which replication is initiated. This can either be DNA replication in living organisms such as prokaryotes and eukaryotes, or RNA replication in RNA viruses, such as double-stranded RNA viruses...
during the initiation step of 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...
. Formation of the pre-RC is required for DNA replication to occur. Complete and faithful replication of the 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....
ensures that each daughter cell will carry the same genetic information as the parent cell. Accordingly, formation of the pre-RC is a very important part of the cell cycle
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
.
Components
As organisms evolved and became increasingly more complex, so did their pre-RCs. The following is a summary of the components of the pre-RC amongst the different domains of life.In prokaryotes, the main component of the pre-RC is DnaA
DnaA
dnaA is a replication initiation factor which promotes the unwinding or denaturation of DNA at oriC , during DNA replication in prokaryotes....
. The pre-RC is complete when DnaA occupies all of its binding sites within the prokaryotic origin of replication (oriC).
The archaeal pre-RC is very different from the prokaryotic pre-RC and can serve as a simplified model of the eukaryotic pre-RC. It is composed of a single origin recognition complex
Origin Recognition Complex
ORC or Origin Recognition Complex is a multi-subunit DNA binding complex that binds in all eukaryotes in an ATP-dependent manner to origins of replication....
(ORC) protein, Cdc6
Cdc6
Cdc6, or Cell Division Cycle 6, is a protein in eukaryotic cells that is studied in the budding yeast Saccharomyces cerevisiae. It is an essential regulator of DNA replication and plays important roles in the activation and maintenance of the checkpoint mechanisms in the cell cycle that coordinate...
, and a homohexamer of the Mini Chromosome Maintenance
Mini Chromosome Maintenance
Mini Chromosome Maintenance complex, or Minichromosome Maintenance protein complex or mini-chromosome maintenance 2-7 helicase complex has a role in both the initiation and the elongation phases of eukaryotic DNA replication, specifically the formation and elongation of the replication fork...
(MCM) protein.
The eukaryotic pre-RC is the most complex and highly regulated pre-RC. In most eukaryotes it is composed of six ORC proteins (ORC1-6), Cdc6, Cdt1, and a heterohexamer of the six MCM proteins (MCM2-7). The MCM heterohexamer arguably arose via MCM gene duplication events and subsequent divergent evolution. The pre-RC of Schizosaccharomyces pombe
Schizosaccharomyces pombe
Schizosaccharomyces pombe, also called "fission yeast", is a species of yeast. It is used as a model organism in molecular and cell biology. It is a unicellular eukaryote, whose cells are rod-shaped. Cells typically measure 3 to 4 micrometres in diameter and 7 to 14 micrometres in length...
(S. pombe) is notably different from that of other eukaryotes; Cdc6 is replaced by the homologous Cdc18 protein. Sap1 is also included in the S. pombe pre-RC because it is required for Cdc18 binding. The pre-RC of Xenopus laevis (X. laevis) also has an additional protein, MCM9, which helps load the MCM heterohexamer onto the origin of replication.
Recognition of the Origin of Replication
Recognition of the origin of replication is a critical first step in the formation of the pre-RC. In different domains of life this process is accomplished differently.In prokaryotes, origin recognition is accomplished by DnaA. DnaA binds tightly to a 9-base pair consensus sequence in oriC; 5' - TTATCCACA - 3'. There are 5 such 9-bp sequences (R1-R5) and 4 non-consensus sequences (I1-I4) within oriC that DnaA binds with differential affinity. DnaA binds R4, R1, and R2 with high affinity and R5, I1, I2, I3, and R3 with lesser affinity. The pre-RC is complete when DnaA occupies all of the high and low affinity 9-bp binding sites
Archaea have 1-3 origins of replication. The origins are generally AT-rich tracts that vary based on the archaeal species. The singular archaeal ORC protein recognizes the AT-rich tracts and binds DNA in an ATP-dependent fashion.
Eukaryotes typically have multiple origins of replication; at least one per chromosome. Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
(S. cerevisiae) is the only known eukaryote with a defined initiation sequence TTTTTATG/ATTTA/T. This initiation sequence is recognized by ORC1-5. ORC6 is not known to bind DNA in S. cerevisiae. Initiation sequences in S. pombe and higher eukaryotes are not well defined. However, the initiation sequences are generally either AT-rich or exhibit bent or curved DNA topology. The ORC4 protein is known to bind the AT-rich portion of the origin of replication in S. pombe using AT hook motifs. The mechanism of origin recognition in higher eukaryotes is not well understood but it is thought that the ORC1-6 proteins depend on unusual DNA topology for binding.
Loading of the Pre-Replication Complex
Assembly of the pre-replication complex only occurs during late M phase and early G1 phaseG1 phase
The G1 phase is a period in the cell cycle during interphase, before the S phase. For many cells, this phase is the major period of cell growth during its lifespan. During this stage new organelles are being synthesized, so the cell requires both structural proteins and enzymes, resulting in great...
of the cell cycle when cyclin-dependent kinase
Cyclin-dependent kinase
thumb|350px|Schematic of the cell cycle. outer ring: I=[[Interphase]], M=[[Mitosis]]; inner ring: M=Mitosis; G1=[[G1 phase|Gap phase 1]]; S=[[S phase|Synthesis]]; G2=[[G2 phase|Gap phase 2]]...
(CDK) activity is low. This timing and other regulatory mechanisms ensure that DNA replication will only occur once per cell cycle. Assembly of the pre-RC relies on prior origin recognition, either by DnaA in prokaryotes or by ORC in archaea and eukaryotes.
The pre-RC of prokaryotes is complete when DnaA occupies all possible binding sites within the oriC.
The pre-RC of archaea requires ORC binding of the origin. After this, Cdc6 and the MCM homohexameric complex bind in a sequential fashion.
Eukaryotes have the most complex pre-RC. After ORC1-6 bind the origin of replication, Cdc6 is recruited. Cdc6 recruits the licensing factor Cdt1 and MCM2-7. Cdt1 binding and ATP hydrolysis by the ORC and Cdc6 load MCM2-7 onto DNA. There is a stoichiometric excess of the MCM proteins over the ORC and Cdc6 proteins, indicating that there may be multiple MCM heterohexamers bound to each origin of replication.
Initiation of Replication
After the pre-RC is formed it must be activated and the replisome assembled in order for DNA replication to occur.In prokaryotes, DnaA hydrolyzes ATP in order to unwind DNA at the oriC. This denatured region is accessible to the 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...
and 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 loader. Single-strand binding protein
Single-strand binding protein
Single-strand binding protein, also known as SSB or SSBP, binds to single stranded regions of DNA to prevent premature annealing. The strands have a natural tendency to revert to the duplex form, but SSB binds to the single strands, keeping them separate and allowing the DNA replication machinery...
s stabilize the newly formed replication bubble and interact with the 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...
. DnaG recruits the replicative 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....
III, and replication begins.
In eukaryotes, MCM heterohexamer is phosphorylated by CDC7 and CDK, which displaces Cdc6 and recruits MCM10
MCM10
Protein MCM10 homolog is a protein that in humans is encoded by the MCM10 gene.-Further reading:...
. MCM10 cooperates with MCM2-7 in the recruitment of Cdc45. Cdc45 then recruits key components 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:...
; the replicative DNA polymerase α and its primase. DNA replication can then begin.
Prevention of Re-Replication
During each cell cycle, it is important that the genome be completely replicated once and only once. Formation of the pre-replication complex during late M and early G1 phase is required for genome replication, but after the genome has been replicated the pre-RC must not form again until the next cell cycle. In S. cerevisiae, CDKs prevent formation of the replication complex during late G1, S, and G2 phases by excluding MCM2-7 from the nucleus, targeting Cdc6 for degradation by the proteasomeProteasome
Proteasomes are very large protein complexes inside all eukaryotes and archaea, and in some bacteria. In eukaryotes, they are located in the nucleus and the cytoplasm. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks...
, and dissociating ORC1-6 from chromatin
Chromatin
Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
via phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
. Metazoans have a fourth mechanism to prevent re-replication; during S and G2 geminin
Geminin
Geminin, DNA replication inhibitor, also known as GMNN, is a protein in humans encoded by the GMNN gene.Geminin is a nuclear protein that is present in most eukaryotics and highly conserved across species...
binds to Cdt1 and inhibits Cdt1 from loading MCM2-7 onto the origin of replication.
Meier-Gorlin Syndrome
Defects in components of the eukaryotic replication complex are known to cause Meier-Gorlin syndrome, which is characterized by absent or hypoplastic patellae, small ears, impaired pre- and post-natal growth, and microcephalyMicrocephaly
Microcephaly is a neurodevelopmental disorder in which the circumference of the head is more than two standard deviations smaller than average for the person's age and sex. Microcephaly may be congenital or it may develop in the first few years of life...
. Known mutations are in the ORC1
ORC1
Origin recognition complex subunit 1 is a protein that in humans is encoded by the ORC1 gene.- Function :The origin recognition complex is a highly conserved six subunits protein complex essential for the initiation of the DNA replication in eukaryotic cells...
, ORC4, ORC6, CDT1, and CDC6 genes. The disease phenotype is probably due to reduced ability of cells to proliferate, reduced cell number, and general growth failure.