We discussed about the Initiation in the Eukaryotic cells in the last post. In the present post, let’s look into the Elongation and the Termination in Eukaryotic DNA Replication.
During Initiation, a repertoire of proteins bind and unwind the DNA at the origin. To this protein complex the polymerases are loaded. The polymerases work together with other proteins for the elongation of the daughter strands.
(Just for info: Read more about the eukaryotic origins in the paper titled ‘Making Sense of Eukaryotic DNA Replication Origins‘.)
Elongation:
The first polymerase to initiate the DNA synthesis is the DNA polymerase α, which exists in the form of DNA polymerase α-primase complex. The primase subunit synthesizes the RNA primers (around 7-12 nucleotides) which are then transferred to the polymerase domain and extended with DNA bases (around 20-25 nucleotides). Replication factor C (RFC) initiates a reaction called polymerase switching. The DNA strands are then extended by other polymerases namely; the DNA polymerase δ and DNA polymerase ε.
(Please read about the lagging strand, leading strands and the Okazaki fragments in a previous post.)
Leading strand synthesis:
As DNA pol α completes synthesizing RNA primer and adding DNA bases, the RFC causes dissociation of DNA pol α and assembles proliferating cell nuclear antigen (PCNA) in the region of the primer terminus. PCNA is a DNA clamp for DNA polymerases.
Pol ε has been reported as the main leading strand synthesis polymerase (in Saccharomyces cerevisiae). In the leading strand as the replication is continuous and the primer is synthesized only once and the extension is carried out (fig 1).
Lagging strand synthesis:
Polymerases δ is the major polymerase in lagging-strand synthesis. The replication in the lagging strands is discontinuous and takes place with formation of several Okazaki fragments (fig 2).
Okazaki fragments (fig 3) generated in eukaryotes during lagging-strand synthesis are around 200 bases (prokaryotes, around 2000 bases) long. As several Okazaki fragments are made, Polymerase switching during synthesis of Okazaki fragments is of higher importance.
Hence an Okazaki fragment is made up of RNA nucleotides (7-10 nucleotides), then around 10-20 nucleotides of DNA bases are added by the DNA pol α. Following which, the RFC causes polymerase switching and the deoxyribonucleotides are added by DNA pol δ held by the PCNA, the sliding clamp (see the figure below). DNA pol δ dissociates after the synthesis of the entire DNA stretch, as it approaches the previous RNA primer.
(Just for info: Know more about PCNA)
RNA primers are removed by RNase H1, such that one ribonucleotide remains still attached to the DNA (3′ end) part of the Okazaki fragment. This left out ribonucleotide is then removed by flap endonuclease 1 (FEN 1). Polymerase δ then fills the gaps formed between Okazaki fragments following the primer removal. The nick formed between the Okazaki fragment and the lagging strand are filled in by DNA ligase I, hence forming single lagging strand.
Nucleosome Assembly:
During elongation, there is continuous disassembly as reassembly of the nucleosome packaging along the DNA, too. As we know, one of the feature of the eukaryotic DNA is that it is packaged in form of highly compact structures called the Chromosomes. It involves winding of the negatively charged DNA around the basic proteins called histones to form structures called nucleosomes (fig below). Each nucleosome is composed of 8 histone proteins, two each of H2A, H2B, H3 and H4. Histone H1 forms the linker between two nucleosomes.
During replication, two nucleosomes in front of the replication fork, that is unreplicated DNA, becomes destabilized. Hence the replication fork movement causes DNA packaging to disorganise to allow the replication proteins to interact with the DNA.
In the replicated portion, the leading and lagging strands were nucleosome-free till about 225 bp and 285 bp, respectively. Following this region, the DNA was packaged with histone octomer but some lacked histone H1. After around 450 bp to 650bp from the replication fork, complete nucleosomes with H1 were detected in the daughter strands. Hence there is stepwise assembly of daughter strands into complete nucleosome.
The parental nucleosomes disassociate and randomly bind the daughter DNA strands, such that each strands has half of the parental nucleosomes. The remaining nucleosome components required for packaging the two daughter strands, are synthesized de novo and assembled onto the daughter strands.
Termination:
Following the successful elongation, the replication has to be terminated to give two separate copies of the DNA.
• Involving two adjacent replication forks:
As the eukaryotic cell have a large number of origins, the termination involves merging of two adjacent replication forks. This includes four different steps:
– Dissolution:
The DNA stretch between the two adjacent forks (fig 5.a) is unwound (fig 5.b) and the approaching CMGs pass each other (fig 5.c). The last Okazaki fragment is processed by DNA pol δ and FEN1 (fig 5.d).
– Ligation:
The gaps in the daughter strands (as in normal Okazaki fragments) are filled in and two oppositely approaching synthesized strands are ligated.
– Dissociation of replisome:
The replisome complex dismantles after the convergence of the two replication fork. This process involves termination-specific polyubiquitylation of Mcm7 and the p97/VCP/Cdc48 segregase (fig 5.e).
– Decatenation:
If there are any intertwinings in the daughter DNA strands or catenanes, they are removed utilising topoisomerase II segregating the two strands.
• Involving the ends of the Chromosomes:
As is known the DNA in eukaryotic chromosomes is a linear molecule, the termination in eukaryotic DNA also involve completing replication at the ends of chromosomes known as Telomeres (fig 6).
During the synthesis of Okazaki fragments, RNA primer provide 3′-OH group for 5′ to 3′ replication. On the removal of RNA primer, from the lagging strand at the chromosome end, the end remains unreplicated and the newly synthesized strand is shortened (fig 7).
(Just for info: Read about the Telomere Banding in a previous post).
This shortening of the chromosome is prevented by the presence of special repeats of sequences called telomeres (and telomere-associated proteins) at the ends of DNA in chromosomes contain. For e.g. human Chromosomes are protected by telomeres having repeated sequences of (TTAGGG)n of about 15–20 kb at birth. These structures protect the ends of chromosomes from being mistakenly considered as DNA double strand breaks (DSB).
In normal somatic cells, the telomeric region of eukaryotic chromosomes are shortened with each round of DNA replication. After certain number of DNA replications, and hence cell divisions, the telomeres are shortened to an extent that it leads to replicative cell senescence or apoptosis.
(Just for info: Please read about the Nobel prize given to a work on telomeres.)
However, in germline and cancer cells, an enzyme called telomerase, extends the ends of the chromosomes, especially the 5′ end of the lagging strands. The ability to maintain the length of the telomeres, make these cells immortal.
Telomerase is a reverse transcriptase which has a RNA template, known as template-encoding RNA molecule (TER) for extension of the telomeric DNA (fig 8). The basic protein component of telomerase is known as TERT (TElomerase Reverse Transcriptase).
In humans, the RNA template has the sequence AUCCCAAUC. The newly synthesized telomeric DNA repeats are added to the overhanging single-stranded 3′ end of the DNA (fig 8).
(Just for info: visit to watch the animation on action of Telomerase and much more.)
After strand extension on the 3′ end by the telomerase is completed, DNA pol α and DNA ligase complete the DNA strand synthesis.
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Suggestions to read:
The Telomere Effect: A Revolutionary Approach to Living Younger, Healthier, Longer (Amazon)
Younger: The Breakthrough Programme to Reset our Genes and Reverse Ageing (Amazon)
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Read more posts by The Biotech Notes:
Bacteriophage Reproduction: Lytic Cycle.
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References:
Bambara et al. (1997) Enzymes and Reactions at the Eukaryotic DNA Replication Fork. J Biol Chem. 272(8):4647-50.
Abmayr and Workman (2012) Holding on through DNA Replication: Histone Modification or Modifier? Cell. 150(5):875-7.
Verhoeven et al (2014). Cellular aging in depression: Permanent imprint or reversible process?: An overview of the current evidence, mechanistic pathways, and targets for interventions. BioEssays 36(10):968-78.
Bailey et al (2015) Termination of DNA replication forks: “Breaking up is hard to do”.Nucleus 6 (3):187-196.
Dewar & Walter (2017) Mechanisms of DNA replication termination. Nature Reviews Molecular Cell Biology 18:507–516.
Aulinas (2013) Telomeres, aging and Cushing’s syndrome: Are they related? Endocrinology and Nutrition (English Edition) 60(6): 329-335.
Boehm et al. (2016) The Many Roles of PCNA in Eukaryotic DNA Replication. Enzymes 39:231-54.
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