Eukaryotic DNA Replication

DNA replication is the process by which an organism makes an exact copy of its genetic material, which it then uses to create offspring. Before a cell divides, its DNA must be replicated so that each daughter cell has an identical set of genetic instructions.

Eukaryotic DNA Replication

• Origins of replication (Ori C) are found in abundance in eukaryotic cells, and replication can proceed in either way.
• Replicating DNA is a semi-conservative process that creates a new copy of the DNA molecule to join the original strand.
• During the S phase, it occurs at numerous chromosomal origins.
• happens inside the nucleus of a cell.
• The only direction in which synthesis can take place is from the fifth to third position.
• There is a leading and a lagging strand of DNA because they are created in opposite directions.
• Okazaki fragments, which are little pieces of DNA, are made and used to create lagging strands.
• The replication process in eukaryotic cells involves five distinct polymerases.

Enzyme Required for Eukaryotic DNA Replication

DNA replication in eukaryotes is very similar to DNA replication in prokaryotes.

There are, however, some differences. Eukaryotic cells are defined by the fact that they can replicate from more than one place. Also, eukaryotes are known to have at least five different DNA polymerases. These enzymes’ numbers are written in Greek letters.

• DNA polymerase α: DNA polymerase  α makes RNA primers for both the leading and the trailing strands of DNA.
• DNA polymerase β: DNA polymerase β is used to fix broken DNA. It does the same thing as DNA polymerase I, which is found in prokaryotes.
• DNA polymerase γ:  DNA polymerase γ helps copy the mitochondrial DNA.
• DNA polymerase G:  DNA polymerase G is in charge of making copies of the DNA on the leading strand. It can also catch mistakes in writing. 

Eukaryotic DNA replication protein list

Function	Bacteria	Eukaryotes
Initiator; binds origin	Dna A	ORC
Helicase loader	DnaC	Cdc6 and Cdt1
Helicase	DnaB	MCM complex
Single-strand binding	SSB proteins	RPA
Primer synthesis	DnaG primase	Pol α-primase1
Replicative DNA polymerase	DNA polymerase III        (C-family polymerase)	DNA polymerase δ and DNA polymerase ε (B-family polymerases)
Clamp loader	ϒ complex	RF-C
Clamp	β clamp	PCNA
Ligase	DNA ligase2	DNA ligase 12
Primer removal	Ribonuclease H; DNA polymerase I	RNaseH/Fen1

Initiation of Eukaryotic DNA replication

1. Origin

• As we saw in the last post, replication starts at a place in the genome called “origin.”
• The DNA sequences at the replication origin stay the same.
• Since the eukaryotic genome is a lot bigger than the prokaryotic genome, it has a lot more origins.
• Because of this, the replication can start at the same time and finish faster.
• Origin has the ORC’s binding sequence.

The origin recognition complex (ORC)

• The origin recognition complex (ORC) is a complex of six subunits organised in a C-shaped structure that encircles the DNA at origin.
• Other elements are bound and recruited into a pre-replicative complex using this complex’s binding and recruitment abilities (pre-RC).

ORC/Cdc6/DNA complex

• In the later stages of the M phase of the cell cycle, a protein known as cell division cycle 6 (Cdc6) binds the origin of replication (ORC)/origin DNA complex.
• For the ORC/Cdc6/DNA complex to develop, ATP binding is essential.

OCCM Complex

• Later, the ORC/Cdc6 complex is bound by the heptamer Cdt1/MCM2-7, which then loads MCM2-7 onto the dsDNA.
• Minichromosome maintenance 2-7 (MCM2-7) is a fundamental component of the replicative DNA helicase and consists of six subunits stacked in a spiral fashion.
• Chromatin licencing and DNA replication factor 1, or Cdt1, is responsible for stabilising and loading a Mcm2-7 hexamer onto the chromosome.
• Cdt1/MCM2-7 binds to the ORC/Cdc6 complex, forming the OCCM complex (fig 1C).

OM intermediate

• Cdc6 and Cdt1 are released from their cellular encapsulation upon creation of this complex, which also triggers the hydrolysis of ATP.
• The ORC/MCM2-7 (OM) intermediate is formed when these two proteins are degraded.

OCM complex

• An additional Cdc6 links to the OM intermediate to form the ORC/Cdc6/MCM2-7 (OCM) complex (fig. 1D), which can then attach a second MCM2-7 hexamer.

DH

• Cdc6, ORC, and Cdt1 are released while the cell is still in G1 phase as a second MCM2-7 hexamer is recruited to the OCM complex and the two hexamers are aligned to form a head-to-head MCM2-7 double hexamer (DH) (fig 1E).

Pre-replication complex

• The creation of the pre-replication complex (pre-RC) is a consequence of the DH’s encirclement of the ds DNA.
• Pre-RC formation might alternatively be thought of as DNA licencing. The RC DH is unable to perform the unwinding actions because it has been dormant since before the RC.

Pre-initiation complex (pre-IC) formation

• The MCM2-7 DHs are phosphorylated and activated by the kinase DDK during the onset of S phase.
• Preinitiation complex (pre-IC) creation describes the assembly of proteins with activated MCM2-7.

CMG complex

• When DH is phosphorylated, a Sld3-binding site on the Mcm2-7 complex becomes accessible. As a result, the Sld3/Sld7 complex and Cdc45 are more likely to attach to replication origins.
• Sld2 and Sld3 are phosphorylated by cyclin-dependent kinase (CDK) during the S phase of the cell cycle. The phosphoproteins Sld2 and Sld3 interact with the DNA replication regulator protein Dpb11, facilitating its recruitment to the origin of replication.

• Mcm2-7 is bound to accessory factors Cdc45 and GINS via a platform formed by Sld2, Sld3, and Dpb11. Cdc45 and GINS interact to produce the Cdc45/MCM2-7/GINS (CMG) complex, which dissociates Sld2, Sld3, and Dpb11.

• Thus, the duplex DNA-encircling MCM2-7 double hexamer undergoes a very controlled transformation into two CMG particles, each of which encircles single-stranded DNA.
• ATP hydrolysis drives 3′-5′ DNA unwinding, or the helicase activity, and the CMG complex associated with another protein called Mcm10 is the primary component at the eukaryotic DNA replication fork (Fig 1G).
• Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that helps stabilise ss DNA as the origin is unwound. Eukaryotic single-stranded DNA binding (SSB) protein is one definition of RPA.
• Eventually, Pol ε is loaded onto the helicase with the aid of the protein ctf4, following a direct interaction between Pol α and the CMG via GINS. Together, Pol α and DNA primase create a four-subunit complex called DNA Polymerase  α-Primase, which is responsible for RNA primer synthesis and subsequent DNA extension. δ
• The pol α- primase complex initiates DNA synthesis, and subsequently polymerase δ and ε polymerase take over for the synthesis of the leading and lagging strands, respectively.

Elongation of Eukaryotic DNA replication

• DNA polymerase α, in the form of DNA polymerase α-primase complex, is the first polymerase to start DNA synthesis.
• Primers, which are short stretches of RNA between 7 and 12 nucleotides in length, are made by the primase subunit and passed to the polymerase domain, where they are extended with DNA bases (around 20-25 nucleotides).
• Polymerase switching is a reaction that is started by replication factor C (RFC). DNA polymerase δ and DNA polymerase ε are two more polymerases that help to lengthen the DNA strands.

Leading strand synthesis

• After DNA pol α has finished synthesising the RNA primer and adding the DNA bases, the RFC dissociates DNA pol α and triggers the assembly of proliferating cell nuclear antigen (PCNA) at the primer terminus region. DNA polymerases use PCNA as a DNA clamp.

• It has been found that Pol  ε  is the primary polymerase involved in leading strand synthesis (in Saccharomyces cerevisiae). Since the extension occurs in the leading strand and replication is continuous, a single round of primer synthesis is sufficient (fig 1).

Lagging strand synthesis

• Polymerases δ is the main polymerase in lagging-strand synthesis. The replication in the lagging strands is discontinuous and takes place with production of numerous Okazaki fragments (fig 2).

• The Okazaki fragments (fig. 3) produced by lagging-strand synthesis in eukaryotes are on the order of 200 bases in length, but in bacteria they are on the order of 2000 bases. Because of the increasing number of Okazaki fragments synthesised, polymerase switching during Okazaki fragment synthesis has taken on more significance.
• Therefore, DNA pol α adds about 10–20 nucleotides of DNA bases to an initial RNA (7–10 nucleotides) in an Okazaki fragment.
• Deoxyribonucleotides are incorporated into strands of DNA by DNA pol δ while the polymerase switching triggered by the RFC is maintained in place by the sliding clamp PCNA (see the figure below). Upon reaching the preceding RNA primer, DNA pol δ dissociates, indicating that DNA synthesis is complete.
• By degrading RNA primers, RNase H1 leaves only one ribonucleotide bound to the DNA (3′ end) of the Okazaki fragment. Flap endonuclease 1 then eliminates the ribonucleotide that was overlooked (FEN 1).

• After the primers are removed, Okazaki fragments are left with gaps in between them, which are filled by polymerase. δ DNA ligase I bridges the nick between the Okazaki fragment and the lagging strand, creating a continuous lagging strand.

Nucleosome Assembly

• The nucleosome packing along the DNA undergoes continual disintegration and reassembly during elongation. One characteristic of eukaryotic DNA is that it is bundled into extremely tiny structures called chromosomes.
• This process involves nucleosome formation, in which negatively charged DNA is wound around basic proteins called histones (fig below).
• There are 8 histone proteins in a nucleosome, 2 each of H2A, H2B, H3, and H4. Between two nucleosomes, histone H1 acts as a linker.
• DNA replication destabilises the two nucleosomes that sit on the strand of unreplicated DNA immediately in front of the replication fork.
• Therefore, DNA packaging becomes disorganised as a result of replication fork movement, allowing replication proteins to interact with the DNA.
• In the duplicated region, nucleosome-free regions extended for around 225 base pairs (bp) and 285 bp (bp) on the leading and lagging strands, respectively. Histone octomer encased the DNA after this area, however part of the chromosomes were missing histone H1.
• Complete nucleosomes containing H1 were found in the daughter strands after approximately 450 bp to 650 bp had passed from the replication fork.
• Therefore, the nucleosome is constructed by the sequential assembly of daughter strands.
• In this process, the parental nucleosomes separate and bind to the daughter DNA strands at random, creating a situation in which each strand receives one-half of the nucleosomes from the other.
• When the two daughter strands are ready for packing, the remaining nucleosome components are generated from scratch and placed onto the strands.

Termination of Eukaryotic DNA Replication

If you want two distinct copies of your DNA, replication must be stopped after it has been completed successfully.

1. Involving two adjacent replication forks

Termination in eukaryotic cells requires the merger of two neighbouring replication forks due to the high number of origins present in these cells. The process entails the following four stages:

Dissolution

• Both CMGs that are moving toward each other unwind the DNA segment between the two forks in Fig. 5.a (Fig. 5.b) (fig 5.c). DNA pol δ and the elongation factor FEN1 work together to process the final Okazaki fragment (fig 5.d).

Ligation

• Two synthesised strands, one approaching from the opposite direction, are ligated together, closing the gaps in the daughter strands (as with regular Okazaki fragments).

Dissociation of replisome

• Following the merger of the two replication forks, the replisome complex is disassembled. Mcm7 and the p97/VCP/Cdc48 segregase are polyubiquitylated at the end of the process (fig 5.e).

Decatenation

• Topoisomerase II is used to separate the two strands of DNA if there are any entanglements, also known as catenanes, in the daughter strands.

2. Involving the ends of the Chromosomes

• It is well known that the DNA in eukaryotic chromosomes is a linear molecule, and that the process of terminating eukaryotic DNA involves completing replication at the ends of chromosomes, which are called Telomeres (fig 6).
• The 3′-OH group from an RNA primer facilitates 5′-to-3′ replication during the creation of Okazaki fragments. After the RNA primer is removed from the unreplicated chromosomal end, the length of the freshly synthesised strand is reduced (fig 7).

• Telomeres (and telomere-associated proteins) are repeating sequences found at the ends of chromosome DNA that protect them from being shortened. Such as a human, At birth, chromosomes have telomeres that are 15-20 kb long and include repeating sequences of (TTAGGG)n. Chromosome ends are safeguarded by these structures to prevent false positives for DNA double strand breaks at their location (DSB).
• When DNA replicates normally in somatic cells, the telomeric region of eukaryotic chromosomes shortens. Replicative senescence or apoptosis occurs when telomeres get too short after a specific number of DNA replications and, thus, cell divisions.
• However, an enzyme called telomerase stretches chromosomal ends, particularly the 5′ end of lagging strands, in germline and cancer cells. These cells are immortal because they are able to keep their telomeres at a constant length.
• Template-encoding RNA molecule (TER) refers to the RNA template used by the reverse transcriptase telomerase (fig 8). TERT is the short form of telomerase RNA-binding protein (TElomerase Reverse Transcriptase).
• Our human RNA template consists of the letters AUCCCAAUC. Telomeric DNA repeats are produced and appended to the 3′ overhang of single-stranded DNA (fig 8).
• DNA pol and DNA ligase finish DNA strand synthesis once telomerase has finished extending the 3′ end of the strand.

DNA Proofreading

Only the elongation-focused polymerases (delta and epsilon) in eukaryotes are capable of proofreading (3′ 5′ exonuclease activity).

As soon as a mistake is found, the incorrect base is deleted using 3′to 5′exonuclease activity and the correct base is inserted in its stead.

Repair by removing and replacing tissue:

Replaces mutant bases, such as pyrimidine dimers, that were created in response to ultraviolet light.

Significance of Eukaryotic DNA Replication

• DNA replication is an elementary mechanism in genetics that is required for cell development and division.
• DNA replication is the process by which a new molecule of the essential nucleic acid DNA is created.
• Regulating cell growth and division depends on DNA replication working as it should.
• As a result, the entire genome is preserved for future generations.

References

• Bhagavan, N. V., & Ha, C.-E. (2015). DNA Replication, Repair, and Mutagenesis. Essentials of Medical Biochemistry, 401–417. doi:10.1016/b978-0-12-416687-5.00022-1 
• Priego, Sara & Gambus, Aga. (2015). Regulation of Unperturbed DNA Replication by Ubiquitylation. Genes. 6. 451-68. 10.3390/genes6030451. 
• Riera et al. (2017) From structure to mechanism- understanding initiation of DNA replication. Genes Dev. 31(11): 1073-1088. doi: 10.1101/gad.298232.117
• Bhagavan & Ha (2015) Chapter 22 – DNA Replication, Repair, and Mutagenesis. Essentials of Medical Biochemistry (Second Edition) With Clinical Cases. 401-417. https://doi.org/10.1016/B978-0-12-416687-5.00022-1
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