DNA replication in eukaryotes

DNA replication in eukaryotes

Eukaryotic genomes are much more complex and larger in size than prokaryotic genomes. The human genome has three billion base pairs per haploid set of chromosomes, and 6 billion base pairs are replicated during the S phase of the cell cycle.

Site of DNA replication: Nucleus

Most replication in the nucleus of a eukaryotic cell takes place at a limited number of fixed sites, often referred to as replication factories.

Eukaryotic cells replicate DNA at the rate of ~ 50 nt/s (~ 20 times slower than does E. coli)

DNA replication is basically similar in Eukaryotes and Prokaryotes

  • Basic mechanisms of DNA replication (including both the geometry of the replication fork and the protein components of the multi protein replication machine) are similar for prokaryotes and eukaryotes.
  • DNA replication follows semi conservative mechanism in both Eukaryotes and Prokaryotes.

The major difference is that eukaryotic DNA is replicated not as bare DNA but as chromatin, in which the DNA is complexed with tightly bound proteins called histones.

Eukaryotic DNA is bound to basic proteins known as histones to form repeating structural units called nucleosomes.

Nucleosomes are spaced at intervals of about 200 base pairs along the DNA, which may be why new Okazaki fragments are synthesized on the lagging strand at intervals of 100 to 200 nucleotides in eukaryotes instead of at intervals of 1000 to 2000 nucleotides as in bacteria.

Nucleosomes may also act as barriers that slow down the movement of DNA polymerase molecules, which could explain why eukaryotic replication forks move only one-tenth as fast as bacterial replication forks.

Before replication can start, the chromatin (the complex between DNA and proteins) may undergo some chemical modifications, so that the DNA may be able to slide off the proteins or be accessible to the enzymes of the DNA replication machinery.

 The other differences are, eukaryotes have:

(1) Multiple replication origins in their chromosomes

(2) More types of DNA polymerases, with different functions

(3) Nucleosome assembly immediately following DNA replication.

Eukaryotes have multiple replicons per chromosome

A segment of DNA whose replication is under the control of one origin and two termini is called a replicon.

  • In prokaryotes, the entire chromosome is usually one replicon.
  • The genomes of humans and other mammals contain about 10,000 origins of replication distributed throughout the chromosomes at 30,000 to 300,000 base pair intervals.
  • There are multiple origins of replication on the eukaryotic chromosome. Humans can have up to 100,000 origins of replication.
  • The number of replicons per chromosome is not fixed throughout the growth and development of a multicellular eukaryote.

(Replication is initiated at more sites during the very rapid cell divisions of embryogenesis than during later stages of development).

Figure 1: bidirectional replication of the multiple replicons in the giant DNA molecules of eukaryotes

Why there are multiple origins of replication in eukaryotes?

  • Prokaryotic chromosomes have one origin of replication, while eukaryotic chromosomes have multiple origins.
  • This is because eukaryotic chromosomes are much larger (typically contains 60 times more DNA than those of prokaryotes) than prokaryotic DNA. Its bidirectional replication from a single origin would require ~1 month for completing replication.
  • In order to duplicate all of the chromosomes efficiently, each eukaryotic chromosome contains one linear molecule of double-stranded DNA having multiple origins of replication.

(Multiple origins are needed to replicate the entire chromosome in a short amount of time).

  • Prokaryotic chromosomes are small, so they can get away with having only one origin.

More number of DNA polymerases with different function

  • A significant difference in the processes of bacterial and eukaryotic replication is in the number and functions of DNA polymerases. Eukaryotic cells contain a number of different DNA polymerases that function in replication, recombination, and DNA repair.
  • The number of DNA polymerases in eukaryotes is much more than prokaryotes. 14 are known, of which five are known to have major roles during replication and have been well studied. They are known as polymerase α, polymerase β, polymerase γ, polymerase δ, and polymerase ε.

 Refer the link for more details

Link: http://easylifescienceworld.com/dna-polymerase-enzyme-dnap-of-eukaryotes/

Nucleosome assembly immediately following replication

  • In the cells of higher eukaryotes, the genomic DNA is of a few metres in length. It is compacted in such a way to be confined within nucleus having only micrometres in diameter. This compaction is brought about by association of the DNA with nuclear proteins like histones.
  • The resulting complex nucleoprotein structure is known as chromatin, and the basic unit of chromatin is the nucleosome core particle.
  • During S phase of the cell division cycle, not only is the entire genomic DNA replicated, but the underlying chromatin structure has to be duplicated as well.
  • Chromatin structure is transiently disrupted during passage of a replication fork to let the replisome duplicate the DNA packaged in them.
  • After that nucleosomes are quickly reassembled. New nucleosomes are subsequently assembled on the emerging DNA daughter strands. (DNA replication and nucleosome assembly are tightly coupled).

Figure 2: Partial disruption of chromatin structure by nucleosome disassembly at the replication fork during DNA replication

Duplication of nucleosomes at replication fork

Since the mass of the histones in nucleosomes is equivalent to that of the DNA, large quantities of histones must be synthesized during each cell generation in order for the nucleosomes to duplicate. Although histone synthesis occurs throughout the cell cycle, there is a burst of histone biosynthesis during S phase that generates enough histones for chromatin duplication.

 Mode of nucleosome duplication: At the protein level, nucleosome duplication appears to occur by a dispersive mechanism.

(The nucleosomes on both progeny DNA molecules were found to contain both old (prereplicative) histone complexes and new (post replicative) complexes).

After replication and nucleosome assembly, do the original histones remain together, attached to one of the new DNA molecules, or do they disassemble and mix with new histones on both DNA molecules?

Answer: Newly assembled octamers consist of a random mixture of old and new histones (Dispersive method)

  • A number of proteins are involved in the disassembly and assembly of nucleosomes during chromosome replication in eukaryotes.
  • Two of the most important are nucleosome assembly protein-1 (Nap-1) and chromatin assembly factor-1 (CAF-1).
  • Nap-1 transports histones from their site of synthesis in the cytoplasm to the nucleus
  •  CAF-1 carries them to the chromosomal sites of nucleosome assembly
  • CAF-1 delivers histones to the sites of DNA replication by binding to PNCA (proliferating cell nuclear antigen)—the clamp that tethers DNA polymerase δ to the DNA template.
Figure 3 and 4: The assembly of new nucleosomes during chromosome replication

When DNA replication does occurs in eukaryotes

  • The division cycle of most eukaryotic cells is divided into four discrete phases: M, G1, S, and G2. M phase (mitosis) is usually followed by cytokinesis.
  • S phase is the period during which DNA replication occurs.
  • In G1 phase of the cell cycle, many of the DNA replication regulatory processes are initiated.
  • During G2 phase, any damaged DNA or replication errors are corrected.

Finally, one copy of the genomes is segregated to each daughter cell at mitosis or M phase. These daughter copies each contain one strand from the parental duplex DNA and one nascent antiparallel strand.

Figure 5: Eukaryotic cell cycle

Eukaryotic replisome

  • Each replisome contains three different polymerases, α, δ, and ε.
  • The DNA polymerase α-DNA primase complex synthesizes the RNA primers and adds short segments of DNA.
  • DNA polymerase δ then completes the synthesis of the Okazaki fragments in the lagging strand, and polymerase ε catalyzes the continuous synthesis of the leading strand.
  • PCNA (proliferating cell nuclear antigen) is equivalent to the β subunit of coli DNA polymerase III (It clamps polymerases δ and ε to the DNA molecule facilitating the synthesis of long DNA chains).
  • RPA (replication protein A) is a eukaryotic single stranded DNA binding protein (counterpart of coli SSB protein) which stabilizes the single stranded DNA generated as a result of unwinding.
  • RFC (replication factor C) is a clamp loader for PCNA which load sliding clamp onto the DNA template.
  • Ribonucleases H1 and FEN-1 (F1 nuclease 1) remove the RNA primers, polymerase δ fills in the gap, and DNA ligase (not shown in the figure) seals the nicks, just as in col
Figure 6: Some of the important components of a replisome in eukaryotes.

Steps in DNA Replication

  1. Identification of the origins of replication
  2. Unwinding (denaturation) of dsDNA to provide an ssDNA template
  3. Formation of the replication fork
  4. Initiation of DNA synthesis

For details refer the following link

Link: http://easylifescienceworld.com/dna-replication-initiation-eukaryotes/

  1. Elongation – Synthesis of leading and lagging strands (Okazaki fragment)
  2. Primer removal and ligation of the newly synthesized DNA segments
  3. Reconstitution of chromatin structure

(Reassembly of nucleosome immediately following replication – already discussed)

Elongation stage in DNA replication

  • During elongation, an enzyme called DNA polymerase adds DNA nucleotides to the 3′ end of the newly synthesized polynucleotide strand.
  • The template strand specifies which of the four DNA nucleotides (A, T, C, or G) is added at each position along the new chain.
  • Only the nucleotide complementary to the template nucleotide at that position is added to the new strand.
  • DNA polymerase contains a groove that allows it to bind to a single-stranded template DNA and travel one nucleotide at a time.

For example, when DNA polymerase meets an adenosine nucleotide on the template strand, it adds a thymidine to the 3′ end of the newly synthesized strand, and then moves to the next nucleotide on the template strand. This process will continue until the DNA polymerase reaches the end of the template strand.

Leading and Lagging strand synthesis

  • DNA polymerase can only synthesize new strands in the 5′ to 3′ direction. Therefore, the two newly-synthesized strands grow in opposite directions because the template strands at each replication fork are antiparallel.
  • The “leading strand” is synthesized continuously toward the replication fork as helicase unwinds the template double-stranded DNA.
  • The leading strand is continuously synthesized by the eukaryotic DNA polymerase δ.
  • The “lagging strand” is synthesized in the direction away from the replication fork and away from the DNA helicase unwinds.
  • Lagging strand is synthesized by DNA polymerase ε.
  • This lagging strand is synthesized in pieces because the DNA polymerase can only synthesize in the 5′ to 3′ direction, and so it constantly encounters the previously-synthesized new strand. The pieces are called Okazaki fragments, and each fragment begins with its own RNA primer.

Polymerase enzyme is not moving along DNA instead it pulls out the DNA template through it

Earlier it was thought that the DNA polymerases that carry out replication are frequently moving down the DNA template as a locomotive travels along a train track. Recent evidence suggests that this view is incorrect. A more accurate view is that the polymerase is fixed in location, and template DNA is threaded through it, with newly synthesized DNA molecules emerging from the other end.

For detailed information on this process, refer the following link

http://easylifescienceworld.com/leading-strand-and-lagging-strand-synthesis-in-e-coli-elongation-stage-of-dna-replication/

Termination of DNA replication

  • Eukaryotes appear to lack termination sequences and proteins analogous to the Ter sites and Tus protein in coli.
  • Eukaryotic chromosomes have multiple origins of replication, which initiate replication almost simultaneously. Each origin of replication forms a bubble of duplicated DNA on either side of the origin of replication.
  • Eventually, the leading strand of one replication bubble reaches the lagging strand of another bubble, and the lagging strand will reach the 5′ end of the previous Okazaki fragment in the same bubble.
  • DNA polymerase halts when it reaches a section of DNA template that has already been replicated.
  • However, DNA polymerase cannot catalyze the formation of a phosphodiester bond between the two segments of the new DNA strand, and it drops off.
  • These unattached sections of the sugar-phosphate backbone in an otherwise full-replicated DNA strand are called nicks.

Once all the template nucleotides have been replicated, the replication process is not yet over. RNA primers need to be replaced with DNA, and nicks in the sugar-phosphate backbone need to be connected.

Primer removal and ligation of Okazaki fragments

  • DNA Polymerases δ and DNA polymerase ε both contain the 3’→ 5’ exonuclease activity required for proofreading. However, they do not have 5’→ 3’ exonuclease activity. Thus, they cannot remove RNA primers like DNA polymerase I of coli does.
  • Instead, the RNA primers are excised by two nucleases (cellular enzymes): RNase H and FEN-1 (Flap endonuclease 1).
  • The enzymes FEN1 and RNase H remove RNA primers at the start of each leading strand and at the start of each Okazaki fragment, leaving gaps of unreplicated template DNA.
  • Once the primers are removed, free-floating DNA polymerase δ land at the 3′ end of the preceding DNA fragment and extend the DNA over the gap (gap filling). However, this creates new nicks (unconnected sugar-phosphate backbone).
  • The Okazaki fragments in the lagging strand are joined together after the replacement of the RNA primers with DNA. The gaps that remain are sealed by DNA ligase, which forms the phosphodiester bond.
  • After ligase has connected all nicks, the new strand is one long continuous DNA strand, and the daughter DNA molecule is complete.

Refer the following link for more details on the action of DNA ligase

Link: http://easylifescienceworld.com/dna-ligase/

  • The two identical chromosomes that results from DNA replication are referred to as sister chromatids.
  • Once the DNA replication has been completed, the sister chromatids remain connected at the centromere.
  • These sister chromatids are divided  equally between daughter cells during mitosis.
Figure 7

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