EUKARYOTIC DNA REPLICATION

 

V. Magendira ManiAssistant Professor, PG & Research Department of Biochemistry,Islamiah College (Autonomous),Vaniyambadi,Vellore District – 6357512,Tamilnadu, India.

magendiramani@rediffmail.com

Also available at https://tvuni.academia.edu/mvinayagam

EUKARYOTIC DNA REPLICATION

DNA replication is the process of producing two

identical replicas from one original DNA molecule.

This biological process occurs in all living organisms

and is the basis for biological inheritance. DNA is

made up of two strands and each strand of the original

DNA molecule serves as template for the production

of the complementary strand, a process referred to as

semiconservative replication. The fundamental

mechanism of eukaryotic replication is same as

prokaryotic DNA Replication but some variation also

there.

The replications in eukaryotes are more complex. Because

DNA molecule of eukaryote

Eukaryotic genomes are quite complex

Considerably larger than bacterial DNA

Organized into complex nucleoprotein structure

(chromatin)

Essential features of DNA replication are the same in

prokaryotes and eukaryotes, Similarities of prokaryotes and

eukaryotic replication

Replication process is fundamentally similar in both

prokaryotes and eukaryotes. Process that are similar Include

Formation of replication fork

Simi conservative replication

Movement of replication fork bidirectional

Primer synthesis

Okazaki fragment synthesis in lagging strand

Primer removal

Gap bridging between newly synthesized DNA

fragments.

Difference between prokaryotic and eukaryotic replication

Overall process of eukaryotic replication is bit more

complex. Important differences are due to

• Larger size of eukaryotic DNA (105-106 Kb) compared to

prokaryotic DNA 15x103 kb in E.Coli

• Distinct package of eukaryotic DNA in the term of

chromatin

• Slower rate of fork movement in eukaryotes

For DNA to become available to DNA polymerase,

nucleotide must dissemble. This step slows the Rate of fork

movement.

Replication rate:

Prokaryotes: An E.Coli replication fork progresses at

approximately 1000 bp / sec.

Eukaryotes: Replication rate ten times slower than

prokaryotes 50 nucleotides / sec.

Enzymes and proteins required for eukaryotic DNA

replication

Eukaryotic DNA polymerase:

In eukaryotes there are five different polymerases and they

differ in

Intracellular compartmentation

Kinetic property

Response to inhibitor

DNA polymerases location function

DNA Pol alpha nucleusDNA replication initiation (both leading

and lagging strand)

DNA Pol Delta nucleus lagging strand synthesis

DNA Pol Epsilon nucleusleading strand synthesis

DNA polymerase Alpha

- Located in nucleus

- Catalysis the initiation of replication on both

leading and lagging strand synthesis

- Tetramer – 4 subunits POLA 1 (catalytic) POLA 1

(regulatory) POLA 3 ,4 (Primase)

- larger subunit - 5´-3´ polymerization activity

-Two smaller subunit – primase activity

- one subunit – assist in other three

subunits

- RNA primer 5-15 nucleotides are subsequently

extended by DNA Pol α.

DNA polymerase Delta

- Located in nucleus

- Catalyzes the synthesis of lagging strand

- Having four subunits – POLD 1,2,3,4

- larger subunits catalyzes 5´-3´ polymerization

activity

- Smaller subunits catalyzes 3´-5´ exonuclease

activity (proof reading activity)

- High processivity when interacting with PCNA

(Proliferating cell nuclear antigen).

PCNA

- Molecular weight 25,000; PCNA is important for both

DNA synthesis and DNA repair

- Multimeric protein

- Found in large amount in nuclei of proliferating

cells.

- Act as “clamp” to keep DNA pol δ from

dissociating off the leading DNA strand. “Clamp”

consist of 3 PCNA molecules each containing two

topologically identical domains that are tightly associated to

form closed ring.

- PCNA helps hold DNA polymerase epsilon (Pol ε)

to DNA.

- DNA pol δ improves fidelity of replication by a

factor of 102 due to its proof reading action. It contributes in

limiting the rates of overall error to 10-9 to 10-12.

- DNA Pol δ is also associated with helicase activity.

DNA polymerase Epsilon - Є

located in nucleus

Having four subunits – POLE 1, (Catalytic)

2,3,4 (subunits)associated with - 5´- 3´ polymerization activity 5’- 3’ exonuclease activity (to remove RNA primer) 3’- 5’ exonuclease activity (to proof read)

DNA pol Є catalyzes the repair mechanism

and catalyzes the removal of primer and

filing the primer gap in Okazaki fragments.

Replicating factor A/ Replicating protein

A (RPA/RFA)

RPA/ RFA are similar to single strand binding

protein. They bind to SS DNA and prevent

the re-annealing of parental DNA.

Replication factor C (RFC)

RFC also called as clamp loader or

match maker.

RFC assist in DNA pol δ to form clamp

between DNA and PCNA.

RFC also plays important role in

setting up a link between DNA pol δ and DNA

pol α, so that the leading strand synthesis and

lagging strand synthesis in eukaryotes can

take place simultaneously.

Histone Dissociation and Association Since DNA is present in packaged form as chromatin, DNA replication is sandwiched between two additional steps in eukaryotes.1. Carefully ordered and in complete dissociation of the chromatin.2. Re-association of DNA with the histone octomers to form nucleosome.Dissociation of histone: methylation at the fifth position of cytosine residues by a DNA methyl transferase appears to functioning by loosening up the chromatin structure. This allows DNA access to proteins and enzymes needed for DNA replication.Synthesis of histone: the synthesis of new histone occurs simultaneously with DNA replication.

SEQUENTIAL STEPS IN EUKARYOTIC DNA REPLICATIONDNA replication is a very complicated process that involves several enzymes and other proteins. It occurs in following stages• Pre-initiation• Initiation• Elongation• Termination• Telomerase function

PRE-INITIATION

Actually during pre-initiation stage, replicator

selection occurs. Replicator selection is the process of

identifying the sequences that will direct the initiation

of replication and occur in G1 phase (prior to S

phase). This process leads to the assembly of a

multiprotein complex at each replicator in the

genome. Origin activation only occurs after cells enter

S phase and triggers the Replicator – associated

protein complex to initiate DNA unwinding and DNA

polymerase recruitment.

Replicator selection is mediated by the formation

of pre-replicative complexes (pre-RCs). The first

step in the formation of the pre-RC is the

recognition of the replicator by the eukaryotic

initiator, ORC (Origin recognition Complex). Once

ORC is bound, it recruits two helicase loading

proteins (cell division cycle protein - Cdc6 and

Cdtl). Together, ORC and the loading proteins

recruit a protein that is thought to be the

eukaryotic replication fork helicase (the Mem 2-7

complex). Formation of the pre-RC does not lead

to the immediate unwinding of origin DNA or the

recruitment of DNA polymerases. Instead the pre-

RCs that are formed during Gl are only activated

to initiate replication after cells pass from the Gl

to the S phase of the cell cycle.

Figure - The steps in the formation of pre-replicative complex (pre-RC)The assembly of the pre-RC is an ordered process that is initiated by the association of the ORC with the replicator. Once bound to the replicator ORC recruits at least two additional proteins Cdc6 and Cdt1 (cell division cycle proteins). These three proteins function together to recruit the putative eukaryotic DNA helicase- the MCM 2-7 (multi-chromatin maintenance protein) complex to complete the formation of the pre-RC

INITIATION

ARS (Autonomously Replicating

Sequences)

In eukaryotes the DNA replication is initiated at

specific site known as ARS

(Autonomously Replication Sequences) or

replicators.

ARS (Origin of chromosome in eukaryotes)

contains

- A central core sequence which contains

highly conserved 11 bp sequence (AT rich

sequence)

- Flanking sequences.

ARS – is 100- 150 long (generally it span about

150 bp)

There are multiple origins in eukaryotes. Eg:

yeast contains 400 ARS. The multiple origins are

spaced 30 -300 kb apart. The sequence between

two origins of replication is called replicons. An

average human chromosome contains as many as

100 replicons and replication may proceed

simultaneously at as many as 200 forks.

- The central core sequence contains 11 bp

elements known as “ARS consensus sequence”

rich in AT pair (It is similar to AT rich 13 mers

present in E.Coli Ori C). It is also called as ORE

(Origin replication element)

- The flanking sequences consist of

overlapping sequence that include varients of core

sequences

ORE (Origin Replicating Element) and

ORC (Origin Recognition Complex)

At the origin there is an association of

sequence specified – ds DNA binding

sequence.

ORE (11 bp sequence in core sequence) binds

to a set of proteins (DNA pol α, DNA pol δ,

RFC, PCNA, RFA, SSB and helicase)

collectively called as ORC Origin Recognition

Complex

ORC is a multimeric protein. Initiation of

replication in all eukaryotes requires this

multimeric subunit protein (ORC) which binds

to several sequences within the replicator.

DUE (DNA Unwinding Element)

ORE located adjacent to approximately 80 bp

AT rich sequence that is easy to unwind.

This is called DUE (DNA Unwinding

Element) Binding of ORC to ORE causes

unwinding at DUE.

Events in replication fork:

When ORC (DNA pol α, DNA pol δ, RFC,

RFA, PCNA, SSB helicase into the origin of

replication especially at ORE, the DNA

synthesis is initiated. The replication fork

moves bi-directionally and replication

proceeds simultaneously as many as 200

forks.

Formation of replication fork:

The replication fork in eukaryotes consists of

four components that form in the following

sequence.

DNA helicase and DNA pol δ (due to its

associated helicase activity) unwinds short

segment of parental DNA at 80 bp AT rich

sequence called DUE (DNA unwinding elements)

which is located adjacent to ORE.

DNA pol α initiated the synthesis of RNA primer.

(DNA pol α is also having primase activity) The

primer is approximately 10 bp.

DNA pol ε in lagging strand and DNA pol δ in

leading strand initiates the daughter strand

synthesis.

SSB and RFA bind to SS DNA and prevent re-

annealing of SS DNA.

In addition to the above, two additional factors play important role in replication of eukaryotesPCNA (proliferating cell nuclear antigen) act as a ‘’clamp’’ to keep DNA pol δ from dissociating off the leading strand and thus increasing the processing of DNA pol ε.RFC also called as ‘clamp loader’ or ‘match maker’.RFC assist in - DNA pol δ to form clamp between DNA and PCNA and - setting up a link between DNA pol δ and DNA pol ε so that the leading Strand and lagging strand synthesis in eukaryotes can take place simultaneously. 

Rate of Replication fork Movement

The rate of replication fork movement in

eukaryote (approximately 50 nucleotide /sec) is

only one tenth that observed in E.Coli at this

rate, replication of an average human

chromosome proceeding from a single origin

would take more than 500 hours. Instead of

that, replication of human chromosome

proceeds bi-directionally from multiple origins

spaced 30-300 kb apart and completed within an

hour.

DNA sequence between two origins of

replication is called replicons. An average

chromosome contains nearly 100 replicons and

thus replication proceeds simultaneously at as

many as 200 forks.

ELONGATION

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. 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.

All newly synthesized polynucleotide strands

must be initiated by a specialized RNA

polymerase called primase. Primase initiates

polynucleotide synthesis and by creating a short

RNA polynucleotide strand complementary to

template DNA strand. This short stretch of RNA

nucleotides is called the primer. Once RNA

primer has been synthesized at the template

DNA, primase exits, and DNA polymerase extends

the new strand with nucleotides complementary

to the template DNA. Eventually, the RNA

nucleotides in the primer are removed and

replaced with DNA nucleotides. Once DNA

replication is finished, the daughter molecules

are made entirely of continuous DNA nucleotides,

with no RNA portions.

The Leading and Lagging Strands

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 "lagging strand" is synthesized in the

direction away from the replication fork and

away from the DNA helicase unwinds. 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.

Leading strand synthesis:- Leading strand synthesis is initiated upon RNA primer, synthesized by the primase subunit of DNA pol α. The RNA primer contains 10-15 nucleotides. - Then DNA pol α adds a stretch of DNA to the primer.- At this point replication factor C (RFC) carries out a process called polymerase switching.- RFC removes DNA pol α and assembles PCNA in the region of primer strand terminus.- Then DNA pol epsilon binds to PCNA and carries out highly processive leading strand synthesis due to its 5’-3’ polymerization activity.- After the addition of several nucleotides in the daughter strand, primer is removed. DNA pol Є due to its 5’-3’ exonuclease activity removes the primer and the gap is filled by the same DNA pol Є due to its 5’-3’ polymerization activity.- Then the nick is sealed by DNA ligase.- DNA pol δ improves the fidelity of replication due

to its proof reading activity.

Lagging strand synthesis:Lagging strand synthesis of Okazaki fragment initiated same way as leading strand synthesis. An Okazaki fragment contains 150-200 nucleotides.RNA primer is synthesised by DNA pol α due to its primase activity.The primer is then extended by DNA pol delta due to its 5’-3’ polymerization activity (lagging strand synthesis), using deoxy ribonucleotides (dNTPs).Priming is a frequent event in lagging strand synthesis with RNA primers placed every 50 or 80 nucleotides.All but one of the ribonucleotides in RNA primer is removed by RNase H1.Then exonuclease activity of FEN 1/ RTH 1 complex removes the one remaining nucleotide. The gap is filled by DNA pol Є by its 5’-3’ polymerase activity.DNA ligase joins the Okazaki fragment of the growing DNA strand.

Combined activity of DNA pol delta and DNA

pol epsilon:-

Looping of lagging strand allows a combined

polymerase delta and polymerase epsilon

asymmetric dimer to assemble and elongate both

leading and lagging strands in the same overall

direction of fork movement.

 

TERMINATION

When the replication forks meet each other, then

termination occurs. It will result in the formation

of two duplex DNA. Even though replication

terminated, 5’ end of telomeric part of the newly

synthesized DNA found to have shorter DNA strand

than the template parent strand. This shortage

corrected by the action of telomerase enzyme and

then only the actual replication completed.

TELOMERES

Eukaryotic chromosomes are linear. The ends

of chromosomes have specialized structures

known as ‘Telomeres’.

Telomeres are – short (5-8 bp)

- tandem repeated and

- GC rich nucleotide

sequence.

- Telomeres form protective cap 7-12 kbp

long in the ends of chromosome. Telomeres

are necessary for chromosome maintenance

and stability. They are responsible for

maintaining chromosome integrity by

protecting against DNA degradation and

rearrangement.

Problem in the completion of replication of

lagging strand:

- Linear genomes including those of

several viruses as well as the chromosomes of

eukaryotic cells force a special problem

completion of replication of the lagging strand.

- Excision of an RNA primer from the 5’

end of a linear molecule would leave a gap

(primer gap). This gap cannot be filled by DNA

polymerase action, because of the absence of a

primer terminus to extend. If the DNA could not

be replicated, the chromosome would shorten a

bit with each round of replication.

- This problem has been solved by

Telomerase.

Telomerase:- Telomerase is ribonucleoprotein. It contains a RNA component which has repeat of 9 to 30 nucleotides long. This RNA component serves as the template for the synthesis of telomeric repeats at the parental DNA ends.- Telomerase is a RNA dependent DNA polymerase with a RNA component.

Telomerase uses the - 3’ end of parental DNA strand as primer,- RNA component of telomerase as template,- adds successive telomeric repeats to the parental DNA strand at its 3’ end due to its 5’-3’ RNA dependent DNA polymerase activity.

Regeneration of telomeres:Telomeric DNA consists of simple tandemly repeated sequences like those shown as below:Telomeric repeats sequence at 5’end -

Organism RepeatHuman AGGGTTHigher plant AGGGTTTAlgae AGGGTTTTprotozoan GGGGTTTTYeast GGGT

These sequences are repeatedly added to the 3’ termini of chromosomal DNAs by ‘Telomerase’. Telomerase uses its RNA component as template and parental DNA as primer. Then by its RNA dependent DNA polymerase activity it repeatedly adds telomeric sequences to the 3’ termini of parental DNA. - Then the telomerase is released.- Finally the RNA primer, (of telomerase) is bound near the lagging strand and it is extended by DNA polymerase. Thus the lagging strand synthesis is completed.

In Linear eukaryotic chromosome, once the first primer on each strand is remove, then it appears that there is no way to fill in the gaps, since DNA cannot be extended in the 3′–>5′ direction and there is no 3′ end upstream available as there would be in a circle DNA. If this were actually the situation, the DNA strand would get shorter every time they replicated and genes would be lost forever.

Elizabeth Blackburn and her colleagues have

provided the answer to fill up the gaps with the

help of enzyme telomerase. So, that the genes

at the ends, are conserved. Telomerase is a

ribonucleoprotein (RNP) i.e. it has RNA with

repetitive sequence. Repetitive sequence

varies depending upon the species example

Tetrahymena thermophilia RNA has AACCCC

sequence and in Oxytrica it has AAAACCCC.

Telomerase otherwise known as modified

Reverse Transcriptase. In human, the RNA

template contains AAUCCC repeats. This

enzyme was also known as telomere terminal

transferase..

.

The 3′-end of the lagging strand template

basepairs with a unique region of the telomerase

associated RNA. Hybridization facilitated by the

match between the sequence at the 3′-end of

telomere and the sequence at the 3′-end of the

RNA. The telomerase catalytic site then adds

deoxy ribonucleotides using RNA molecule as a

template, this reverse transcription proceeds to

position 35 of the RNA template. Telomerase then

translocates to the new 3′-end by pairing with

RNA template and it continues reverse

transcription. When the G-rich strand sufficiently

long, Primase can make an RNA primer,

complementary to the 3′-end of the telomere’s G-

rich strand. DNA polymerase uses the newly

made primer to prime synthesis of DNA to fill in

the remaining gap on the progeny DNA. The

primer is removed and the nick between

fragments sealed by DNA ligase

V. Magendira ManiAssistant Professor, PG & Research Department of Biochemistry,Islamiah College (Autonomous),Vaniyambadi,Vellore District – 6357512,Tamilnadu, India.magendiramani@rediffmail.com ; vinayagam magendiramani@academia.edu

https://tvuni.academia.edu/mvinayagam