presentation on eukaryotic dna replication

SEMINAR ON EUKARYOTIC DNA REPLICATION

SUBMITTED BY – DEVENDRA UPRETI

M.SC. II yr

DefinitionDefinition“The process of making an identical copy of a

duplex (double-stranded) DNA, using existing DNA as a template for the synthesis of new DNA strands.”

• DNA replication at heterochromatin region and telomeric region is slow while at euchromatin region and centromeric dna replication is faster.

• Human 3.0 x 106 • E. coli 4.5 x 103

• Drosophila 1.7 x 105

• Maize 2.0 x 106

Chromosomes in Eukaryotic cells consist of:

DNAproteinsome

chromosomal RNA

G1 preparing for DNA

replication (cell growth)

SDNA replication

G2a short gap before

mitosisM

mitosis and cell division

Cell cycle

RNA

PROTEIN

DNA

Central Dogma

Central Dogma

Semiconservative replication

Conservative replication

Dispersive replication

Experiment of DNA semiconservative replication

"Heavy" DNA(15N)

grow in 14N medium

The first generation

grow in 14N medium

The second generation

DIFFERENCESDIFFERENCES

feature Prokaryote Eukaryote

location Occurs inside the cytoplasm Occurs inside the nucleus

ori one origin of replication per molecule of DNA

Have many origins of replication in each chromosome

Ori length 100-200 or more nucleotides in length

about 150 nucleotides

Initiation element carried out by protein DnaA and DnaB

carried out by the Origin Recognition Complex

DNA polymerase I,II,III α,β,γ,δ,ε

Nucleotide length of Okazaki

1000-2000 nucleotides 200 nucleotides

Basic Basic rulesrules of replication of replication

• Semi-conservative• Starts at the ‘origin’

• Semi-discontinuous

• Synthesis always in the 5'--> 3' direction

• RNA primers required• In humans occurs at the rate of 3000

nucleotide/minute.• In bacterial cell it occurs at the rate of 30,000

nucleotide/minute.

DNA Replication machinery involved in Eukaryotes

• DNA Polymerases α,δ,ε,γ.• Helicase• Topoisomerase

• Primase• Ligase• SSB Proteins• dNTPs, Mg+2, & ATP

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DNA Polymerase α• Involved in initiation

• Synthesizes an RNA primer then adds dNTPs

• A complex of four subunits 50-kD and 60-kD are primase subunits;180-kD subunit DNA polymerase, Synthesizes 10-12 nt RNA primers.

• synthesizes RNA primers for the leading strands and each lagging strand fragments.

DNA Polymerase δ

• The principal DNA polymerase ,add okazaki fragments in lagging strand.

• Has role in proofreading.

• Consists of a 125 kD and a ~50 kD subunit.

• The 50 kd subunit interacts with PCNA (Proliferating Cell Nuclear Antigen).

DNA Polymerase ε:

• Catalyze replication of leading strand ,also contribute in proofreading.

• DNA Polymerase ε Consist of more than one subunit, >300 kd

• Polymerases δ and ε both contain th 3‘ 5'exonuclease activity required for proof reding.

DNA Polymerase β:• Monomeric having 36-38 kd Involved in DNA repair

process.

DNA Polymerase γ:

• The DNA-replicating enzyme of mitochondria

Proteins Involved in Eukaryotic DNA Synthesis:

PCNA (Proliferating Cell Nuclear Antigen)

Provide substrate for DNA Polymerase δ, the eukaryotic counterpart of the Sliding Clamp of E. coli PCNA also encircles the double helix.

RPA (Replication Protein A)

ssDNA-binding protein that facilitates the unwinding of the helix to create two replication forks ,the eukaryotic counterpart of the SSB protein.

RFC (Replication Factor C)

The eukaryotic counterpart of the complex Clamp Loader of E. coli that is it loads PCNA on DNA .

ORC ( Origin recognition complex)

Multisubunit protein, binds to sequences within replicator Interacts with two other proteins – CDC6 & CDT1 resulting in loading of MCM complex on DNA .

MCM (Mini chromosome maintenance complex) Heterohexamer(MCM2-MCM7), ring shaped replicative helicase, Once the Mcm proteins have been loaded onto the chromatin, ORC and Cdc6 can be removed from there.

.

DNA ligase

• They are class of enzymes that catalyze the formation of alpha phosphodiester bond between two DNA chains.

• This enzyme bind OH group at the 3' end of DNA strand to phosphate group at 5' end of the other.

• linking the two fragments and completing replication of the region of the lagging strand.

STAGES

Initiation

Elongation

Termination

– During initiation, proteins bind to the origin of replication while helicase unwinds the DNA helix and two replication forks are formed at the origin of replication.

– During elongation, a primer sequence is added with complementary RNA nucleotides, which are then replaced by DNA nucleotides.

– During elongation the leading strand is made continuously, while the lagging strand is made in pieces called Okazaki fragments.

– During termination, primers are removed and replaced with new DNA nucleotides and the backbone is sealed by DNA ligase.

initiation

To accomplish this, initiation is partitioned into two temporally discrete steps:

•Pre replication complex formation during G1 phase

•Activation of pre RC by cdk and ddk enzymes in s phase.

Cell Cycle Regulation

• G1 phase of the cell cycle there are low levels of CDK activity.

• This allows for the formation of new pre-RC complexes

• During the remaining phases of the cell cycle there are elevated levels of CDK activity.

• This inhibiting new pre-RC complex formation.

• At the transition of the G1 stage to the S phase of the cell cycle, S phase–specific cyclin-dependent protein kinase (CDK) and Dbf4 kinase (DDK) transform the pre-RC into an active replication fork.

Effect of Cdk Activity on pre-RC Formation and Activation

Step in the Formation of the pre-RC• Recognition of the replicator by the

eukaryotic initiator, ORC (Origin recognition Complex)

• Once ORC is bound, it recruits two helicase loading proteins Cdc6 (cell division cycle 6 protein) and cdt1 (chromatin licensing and DNA replication factor 1 protein).

• ORC and the loading proteins recruite eukaryotic replication protein i.e. helicase (the Mcm 2-7 complex)

• Instead the pre-RCs that are formed during G1 are only activated to initiate replication after cells pass from the G1 to the S phase of the cell cycle.

• Phosphorylation of these proteins results in the assembly to add additional replication proteins at the origin and the initiation of replication at S phase.

• These new proteins include the three eukaryotic DNA polymerases.

• Then converted to the active CMG(Cdc45-MCM-GINS) helicase during S phase.

• OnceDNA Polα/ primase synthesizes an RNA primer and briefly extends it. Thus initiation of replication started.

• RFC ( replication factor c) recognizes primer-template junctions and loads PCNA ( proliferating cell nuclear antigen) at these sites.

Enzymatic activity• Topoisomerases are responsible for removing supercoils ahead of the replication fork.

• Helicase unwind the strands at fork.

• Pol α is associated with an RNA primase synthesizing a primer of 10 nucleotide stretch of RNA.

• Pol α for priming synthesis & elongates the newly formed primer with DNA nucleotides.

The Eukaryotic Replication forkThe Eukaryotic Replication fork::

• The progress of the replication fork in eukaryotes is maintain by helicase activity .

• The separated polynucleotides are prevented from reattaching by SSBs Proteins.

• The main SSBs Protein in eukaryotes is RPA

Leading strand synthesis• Starts with the primase activity of DNA Pol α to lays down an RNA

primer, then the DNA pol component of Pol α adds a stretch of DNA.

• RFC assembles PCNA at the end of the primer, PCNA displaces DNA Pol α.

• DNA polymerase δ or e binds to PCNA at the 3’ ends of the growing strand to carry out highly processive DNA synthesis Leading strand synthesis.

• The DNA Polymerase α can extend the initial RNA primer with about 20 nucleotides of DNA but not capable of lengthy DNA synthesis.

• After that DNA polymerase δ recognizes this primer and begins

leading strand synthesis in 5′ —> 3′ direction,

Priming DNA Synthesis in Bacteria & Eukaryotes

Lagging strand synthesisLagging strand synthesis

• RNA primers synthesized by DNA polymerase α. in the 5′ 3′ direction.

• DNA polymerase d will synthesize short fragments of DNA called Okazaki fragments which are added to the 3' end of the primer.

• Completion of lagging-strand synthesis requires removal of the RNA primer from each okazaki fragments.

• To solve this problem there is two models for completion of lagging- strand synthesis in eukaryotes are describe:

• The RNase H model

• The flap model

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Rnase H modelffFlap model

• Histone syntesis is continuous threw out the cell cycle but most amount produced during s phase.

• protein responsible for that are NAP-1 (nucleosome assembly protein) having a role in to transport histone from cytoplasm to nucleus.

• other protein is CAF-1(chromatin assembly factor) that deliver histone to the site of replication .

HOW REPLICATION HAS BEEN TAKE PLACE AT TELOMERIC ENDS....?

TERMINATION

• DNA polymarase DNA polymarase can not replicate the end of can not replicate the end of lagging strand because polymerase requireslagging strand because polymerase requires 3' OH OH end to bind at daughter strand. so it require some end to bind at daughter strand. so it require some additional enzyme to perform it.additional enzyme to perform it.

• TELOMERETELOMERE is G - C rich terminal portion of is G - C rich terminal portion of chromosome.chromosome.

• Highly repetative and conserved sequenced present Highly repetative and conserved sequenced present in eukaryotes.in eukaryotes.

• TELOMERIC sequence in -TELOMERIC sequence in - MAMMALS- TTAGGGMAMMALS- TTAGGG ARABIDOPSIS- TTTAGGGARABIDOPSIS- TTTAGGG PARAMECIUM-TTGGGGPARAMECIUM-TTGGGG

Termination of Replication in Eukaryotes• Removal of the terminal primer at the end of lagging-strand

synthesis leaves a smallgap that cannot be filled in, if left unfixed, this gap leads to the loss of terminal sequences.This is known as the end-replication problem. Telomerase (telomere terminal transferase) is a ribonucleoprotein solve this problem

• Telomerase contains protein and RNA which is complementary to the DNA sequence found in the telomeric repeat. Telomerase extend the 3' end of the parental chromosome beyond the 5‘ end of the daughter strand.

• Telomeric repeat-binding factor (TRF1) and TRF2 bind to double-stranded telomeric DNA and have a role in telomere stabilization and telomere-length regulation. The protruding 3′ end can invade the duplex DNA and form a lariat-like structure called ‘t-loop’, establishing a protective cap .

Telomeric activity and agingTelomeric activity and aging

• Telomerase activity is repressed in the somatic cells of multicellular organism, resulting in a gradual shortening of the chromosomes with each cell generation, and ultimately cell death (related to cell aging).

• This telomerase activity find in germ cells and absent in somatic cell, except cancerous cell.

• In humans due to shortening of telomeric length cause a inherited diseases of premature aging known as progerious occur in teen age.

Fast & accurate!• It takes E. coli <1 hour to copy

5 million base pairs in its single chromosome .

• Human cell copies its 6 billion bases & divide into daughter cells in only few hours with remarkably accuracy.

• DNA polymerase initially makes about 1 in 10,000 base pairing errors .

• Enzymes proofread and correct these mistakes .

• The new error rate for DNA that has been proofread is 1 in 1 billion base pairing errors .

Check points.Check points.

• In order to preserve genetic information during cell division, DNA replication must be completed with high fidelity.

• To achieve this task, eukaryotic cells have proteins in place during certain points in the replication process that are able to detect any errors during DNA replication and are able to preserve genomic integrity.

• These checkpoint proteins inactivate cyclin-dependent kinases to inhibit cell cycle progression from G1 to S.

Gene Protein Function

ATMataxia-telangiectasia mutated

response to DNA damage & arrest cell cycle

ATR ataxia- and Rad3-related Arrest cell cycle

PRKDCDNA-dependent protein kinase catalytic subunit (DNA-PKcs)

Arrest cell cycle

Telomeric replication

• Telomeres are special nucleoprotein structures composed of double-stranded (TTAGGG)n DNA repetitive sequence ranging from 3 to 15 kb ∼and a number of telomere associated proteins.

• The ds telomeric DNA terminates at a 3′ single-stranded G-rich overhang of about 12–500 bases. This protruding 3′ end can invade the duplex DNA and form a lariat-like structure called ‘t-loop’.Establishing a protective cap that shields chromosome ends from being recognized as damaged DNA and prevents nucleolytic degradation and inappropriate fusions of telomeres.

• The t-loop is stabilized by a complex of ds and ss stranded telomere binding proteins known as the ‘shelterin’ proteins [TRF1, TRF2]. The primary role of shelterin is to mark telomeres as the natural chromosome ends and suppress the DNA damage response pathways at telomeres

Editing & proofreading DNA

• DNA polymerase δ and ε• proofreads & corrects mistakes

• repairs mismatched bases

• removes abnormal bases

• repairs damage throughout life

Proofreading refers to any mechanism for correcting errors in protein or nucleic acid synthesis that involves scrutiny of individual units after they have been added to the chain

conclusion

• The process of DNA replication is highly conserved throughout evolution

• Major replication features in simpler organisms extend uniformly to eukaryotic organisms

• Thus eukaryotes replicate their DNA in semi conservative manner

• So, the complete and accurate DNA replication is integral to the maintenance of the genetic integrity of all organisms.

References

• Greider, C.W. 1996. Telomere length regulation. Annu. Rev.Biochem. 65:337–365.

• • Rudolph, K.L., S. Chang, H.-W. Lee, M. Blasco, G.J. Gottlieb,C. Greider,

and R.A. DePinho. 1999. Longevity,stressresponse, and cancer in aging telomerase-deficient mice. Cell.96:701–712.

• M. & McBride, H. Rab,2001, proteins as membraneorganizers. Nature Rev. Mol. Cell Biol. 2, 107–117.

• D. PETER SNUSTAD, MICHAEL J. SIMMONS,2012 ,Genetics,Jhon wiley & sons Singapore Pte Ltd, 10,244-249.

• BUCHANAN , GRUISSEM , JONES ,2007, Biochemistry & molecular bilogy of plants. I.K.International Pvt. Ltd, 06, 260-277.

• Tomaska, L., Makhov, A. M., Griffith, J. D. & Nosek. 2002, J. t-loops in yeast mitochondria. Mitochondrion 1, 455–459 .

• Anja-Katrin Bielinsky and Susan A. Gerbi . Journal of Cell Science The Company of Biologists Ltd, 114, 643-651.

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