Eukaryotic DNA replication
Encyclopedia
DNA replication
in eukaryotes is much more complicated than in prokaryotes, although there are many similar aspects. Eukaryotic cells can only initiate DNA replication at a specific point in the cell cycle, the beginning of S phase
.
. However, pre-initiation occurs in the G1 phase
. Thus, pre-initiation and activation require that two very different intra-cellular contexts follow each other in the right order, making it very unlikely that replication take place more than once per cell cycle.
Due to the sheer size of chromosomes in eukaryotes, eukaryotic chromosomes contain multiple origins of replication
. Some origins are well characterized, such as the autonomously replicating sequences (ARS) of yeast while other eukaryotic origins, particularly those in metazoa, can be found in spans of thousands of basepairs. However, the assembly and initiation of replicaton is similar in both the protozoa
and metazoa.
(the pre-RC). The formation of this complex occurs in two stages. The first stage requires that there is no CDK
activity. This can only occur in early G1. The formation of the pre-RC is known as licensing, but a licensed pre-RC cannot initiate replication in the G1 phase
Current models hold that replication begins with the binding of the origin recognition complex (ORC) to the origin. This complex is a hexamer of related proteins and remains bound to the origin, even after DNA replication occurs. Furthermore, ORC is the functional analogue of prokaryotic DnaA
. Following the binding of ORC to the origin, Cdc6
/Cdc18 and Cdt1 coordinate the loading of the MCM (Mini Chromosome Maintenance
) complex to the origin by first binding to ORC and then binding to the MCM complex. The MCM complex is thought to be the major DNA helicase in eukaryotic organisms. Once binding of MCM occurs, a fully licensed pre-RC exists.
and Ddk. The activation process begins with the addition of Mcm10
to the pre-RC, which displaces Cdt1. Following this, Ddk phosphorylates Mcm3-7, which activates the helicase. It is believed that ORC and Cdc6/18 are phosphorylated by Cdk2-Cyclin E. Ddk and the Cdk complex then recruits another protein called Cdc45, which then recruits all of the DNA replication proteins to the replication fork. At this stage the origin fires and DNA synthesis begins.
Activation of a new round of replication is prevented through the actions of the cyclin dependent kinases and a protein known as geminin
. Geminin binds to Cdt1 and sequesters it. It is a periodic protein that first appears in S-phase and is degraded in late M-phase, possibly through the action of the anaphase promoting complex (APC). In addition, phosphorylation of Cdc6/18 prevent it from binding to the ORC (thus inhibiting loading of the MCM complex) while the phosphorylation of ORC remains unclear. Cells in the G0 stage of the cell cycle are prevented from initiating a round of replication because the Mcm proteins are not expressed.
At least three different types of eukaryotic DNA polymerase
s are involved in the replication of DNA in animal cells (POL α, Pol δ and POL ε).
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...
in eukaryotes is much more complicated than in prokaryotes, although there are many similar aspects. Eukaryotic cells can only initiate DNA replication at a specific point in the cell cycle, the beginning of S phase
S phase
S-phase is the part of the cell cycle in which DNA is replicated, occurring between G1 phase and G2 phase. Precise and accurate DNA replication is necessary to prevent genetic abnormalities which often lead to cell death or disease. Due to the importance, the regulatory pathways that govern this...
.
Location in cell cycle
DNA replication in eukaryotes occurs only in the S phase of the cell cycleCell 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...
. However, pre-initiation occurs in the G1 phase
G1 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...
. Thus, pre-initiation and activation require that two very different intra-cellular contexts follow each other in the right order, making it very unlikely that replication take place more than once per cell cycle.
Due to the sheer size of chromosomes in eukaryotes, eukaryotic chromosomes contain multiple origins 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...
. Some origins are well characterized, such as the autonomously replicating sequences (ARS) of yeast while other eukaryotic origins, particularly those in metazoa, can be found in spans of thousands of basepairs. However, the assembly and initiation of replicaton is similar in both the protozoa
Protozoa
Protozoa are a diverse group of single-cells eukaryotic organisms, many of which are motile. Throughout history, protozoa have been defined as single-cell protists with animal-like behavior, e.g., movement...
and metazoa.
Preparation in G1 phase
The first step in DNA replication is the formation of the pre-initiation replication complexPre-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...
(the pre-RC). The formation of this complex occurs in two stages. The first stage requires that there is no CDK
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]]...
activity. This can only occur in early G1. The formation of the pre-RC is known as licensing, but a licensed pre-RC cannot initiate replication in the G1 phase
Current models hold that replication begins with the binding of the origin recognition complex (ORC) to the origin. This complex is a hexamer of related proteins and remains bound to the origin, even after DNA replication occurs. Furthermore, ORC is the functional analogue of prokaryotic DnaA
DnaA
dnaA is a replication initiation factor which promotes the unwinding or denaturation of DNA at oriC , during DNA replication in prokaryotes....
. Following the binding of ORC to the origin, 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...
/Cdc18 and Cdt1 coordinate the loading of the MCM (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...
) complex to the origin by first binding to ORC and then binding to the MCM complex. The MCM complex is thought to be the major DNA helicase in eukaryotic organisms. Once binding of MCM occurs, a fully licensed pre-RC exists.
Synthesis in S phase
Activation of the complex occurs in S-phase and requires Cdk2-Cyclin ECyclin E
Cyclin E is a member of the cyclin family.Cyclin E binds to G1 phase Cdk2, which is required for the transition from G1 to S phase. The Cyclin E/CDK2 complex phosphorylates p27Kip1 , tagging it for degradation, thus promoting expression of Cyclin A, allowing progression to S phase....
and Ddk. The activation process begins with the addition of Mcm10
MCM10
Protein MCM10 homolog is a protein that in humans is encoded by the MCM10 gene.-Further reading:...
to the pre-RC, which displaces Cdt1. Following this, Ddk phosphorylates Mcm3-7, which activates the helicase. It is believed that ORC and Cdc6/18 are phosphorylated by Cdk2-Cyclin E. Ddk and the Cdk complex then recruits another protein called Cdc45, which then recruits all of the DNA replication proteins to the replication fork. At this stage the origin fires and DNA synthesis begins.
Activation of a new round of replication is prevented through the actions of the cyclin dependent kinases and a protein known as 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...
. Geminin binds to Cdt1 and sequesters it. It is a periodic protein that first appears in S-phase and is degraded in late M-phase, possibly through the action of the anaphase promoting complex (APC). In addition, phosphorylation of Cdc6/18 prevent it from binding to the ORC (thus inhibiting loading of the MCM complex) while the phosphorylation of ORC remains unclear. Cells in the G0 stage of the cell cycle are prevented from initiating a round of replication because the Mcm proteins are not expressed.
At least three different types of eukaryotic DNA polymerase
Eukaryotic DNA polymerase
There are at least 15 Eukaryotic DNA polymerases:*POLA1, POLA2: Pol α : forms a complex with a small catalytic and a large noncatalytic subunit, with the Pri subunits acting as a primase , and then with DNA Pol α elongating that primer with DNA nucleotides...
s are involved in the replication of DNA in animal cells (POL α, Pol δ and POL ε).
- Pol α forms a complex with a small catalytic (PriS) and a large noncatalytic (PriL) subunit, with the Pri subunits acting as a primase (synthesizing an RNA primer), and then with DNA Pol α elongating that primer with DNA nucleotides. After around 20 nucleotides elongation is taken over by Pol ε (on the leading strand) and δ (on the lagging strand).
- Pol δ: Highly processive and has proofreading 3'->5' exonuclease activity. Thought to be the main polymerase involved in lagging strand synthesis, though there is still debate about its role.
- Pol ε: Also highly processive and has proofreading 3'->5' exonuclease activity. Highly related to pol δ, and thought to be the main polymerase involved in leading strand synthesis, though there is again still debate about its role.