DNA Replication in Eukaryotes

Views:
 
     
 

Presentation Description

Eukaryotic DNA replication .

Comments

By: vaijayantiMogal (2 month(s) ago)

vaijayantigunjal allow to download

By: nuhaajram9 (3 month(s) ago)

nice and easier ppt

By: areshazain3 (4 month(s) ago)

nice presntation sir can u send me....

By: rlravikumar (12 month(s) ago)

allow down load

Presentation Transcript

Slide 1:

Replication Of dna In eukaryotes By : Dr Mansoor Nabi Mir

Slide 2:

DNA replication in eukaryotes Similar to that in prokaryotes: 1) Similar replication fork geometry 2) Using analogous multiprotein replication machinery

Origin of replication:

Origin of replication Eukaryotic chromosomes contain multiple replication origins. This is necessary because: Eukaryotes have more DNA to replicate Eukaryotic DNA polymerase is slower than prokaryotic Rate of DNA synthesis in eukaryotes is 50NTs/sec as compaired to about 1000 NTs/sec in prokaryotes. If single origin = 20 days (Normal 7 hours)

Eukaryotes have Multiple Origins of Replication:

Eukaryotes have Multiple Origins of Replication After every 1-300 kbp of DNA to make sure that DNA replication occurs continuously throughout the chromosome. Depending on the organism there is a replication origin or “replicator” In lower eukaryotes such as yeast replicator sequences (autonomously replicating sequences ARS ) have been found and they comprise 100-200 bp In mammalian chromosomes no specific sequences for origin.The zones where initiation of replication occurs can span 500-50,000 bp.

Slide 5:

Multiple origins of replication in Eukaryotes +

Slide 6:

Species Genome Speed S phase No of chromosomes(haploid) Origins E. coli 4.6 Mbp 30 kb/min 40 Mins 1 1 Yeast 14Mbp 3kb/min 20 Mins 16 330 mouse 2.5Bbp 2.2kbp/min - 20 25000 Fruit fly 180Mbp 2.6kbp - 9 3500 Human 3.2Bbp 3kb/min 7 Hrs 23 >10000 ? Genome size and rate of DNA synthesis

Slide 7:

Eukaryotic DNA Polymerases Multiple DNA polymerases (at least 13) have been identified in eukaryotes α and δ – are essential for DNA replication β and ε – DNA repair γ – mtDNA synthesis Other types – κ , η , τ , etc. ( Role yet to be elucidated ) Enzymes involved in eukaryotic DNA replication

Slide 8:

DNA Polymerase α Involved in initiation A complex of four subunits 50-kD and 60-kD are primase subunits;180-kD subunit DNA polymerase Synthesizes 10-12 nt RNA primers, then adds DNA to the RNA primers Low processivity of DNA synthesis Has no 3’ -5’ exonuclease activity (proofreading), yet has high fidelity

Slide 9:

DNA Polymerase δ The principal DNA polymerase in eukaryotic DNA replication Has 3’-5’ exonuclease activity Has two subunits, one 125 kd and other ~50 kd The 50 kdal subunit interacts with PCNA (Proliferating Cell Nuclear Antigen) Is highly processive when in association with PCNA

Slide 10:

DNA Polymerase β Monomeric having 36-38 kd Involved in DNA repair process DNA Polymerase ε Consist of more than one subunit, >300 kd Role in DNA repair (doesn’t participate in replication) May be involved in the removal of primer of Okazaki’s fragments DNA Polymerase γ Has 2 subunits Present in mitochondria mtDNA Synthesis(polymerase as well as exonuclease activity)

Proteins Involved in Eukaryotic DNA Synthesis:

Proteins Involved in Eukaryotic DNA Synthesis PCNA (Proliferating Cell Nuclear Antigen) Confers high processivity to DNA Polymerase δ The eukaryotic counterpart of the 2 Sliding Clamp of E. coli PCNA also encircles the double helix, but is a homotrimer of 37 kD subunits 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 of E. coli

Slide 13:

RFC (Replication Factor C) The eukaryotic counterpart of the complex Clamp Loader of E. coli that is it loads PCNA on DNA MCM (Mini chromosome maintenance) complex Heterohexamer(MCM2-MCM7), ring shaped replicative helicase 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 strand

Slide 14:

Post-replicative state Ori Pre-RC (Pre-Replicative Complex) Assembly of thé prereplicative complex & Triggering of Replication 1 ORC MCMs Noc3 Cdc45 Cdc6 Cdt1 S M G 1 G 2 Pre-RC assembly Cdk1 Dbf4 Cdc7 SPF (S-phase Promoting Factor) S-CDK DDK 2 Initiation of DNA replication DDK S-CDK Pre-RC disassembly

Slide 15:

Regulation of initiation of replication

Slide 16:

Analogous DNA replication Factors Function E. coli Eukaryotes Helicase DnaB MCM Complex 2.Relax torsional strain DNA gyrase Topoisomerase II 3. ssDNA binding SSB RPA 4. Primase DnaG POL /primase 5. Primary replicating POL III POL  polymerase 6.Sliding clamp  subunit PCNA 6. Clamp loader  complex RFC 7. Excision of primer Pol I RNaseH1 and FENI 8. Gap filling pol I pol  (and/or pol ?) 9. Nick sealing DNA ligase DNA ligase (NAD dep.) (ATP dep)

Slide 17:

Starts with the primase activity of DNA Pol α to lay down a primer 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 5) DNA polymerase δ binds to PCNA at the 3’ ends of the growing to carry out highly processive DNA synthesis Leading strand synthesis

Lagging strand synthesis:

Lagging strand synthesis RNA primers synthesized by DNA polymerase a every 100 nt and consist of 10 nt RNA + 10-20 nt DNA Polymerase switching as before to extend the RNA-DNA primers to generate Okazaki fragments When the DNA Pol δ approaches the RNA primer of the downstream Okazaki fragment, RNase H1 removes all except last RNA nucleotide of the RNA primer (which is removed by FEN 1) DNA Pol δ fills in the gap as the RNA primer is being removed DNA ligase joins the Okazaki fragment to the growing strand

Slide 19:

Synthesis of leading and lagging strands MCM (Helicase) DNA Pol δ DNA Pol α RFC (Clamp loader) PCNA (Clamp) RPA Leading strand Lagging strand

Slide 20:

RNase H1 Primer FEN 1 PCNA Primer removal

Slide 21:

Terrmination of replication Every time a linear chromosome replicates, the lagging strand at each end gets shorter by about 150 nts. because there is a minimum length of DNA needed for initiation of an Okazaki fragment. This is termed the “end-replication problem”.

Slide 22:

Telomeres The ends of eukaryotic chromosomes are called telomeres (chromosomes are linear dsDNA molecules) Telomeres are needed for chromosomal integrity and stability (protect ends from degradation).

Slide 23:

Facts about Telomeres Somatic cells lack telomerase activity because the TRT gene is switched off Therefore, the telomeres get shorter with each cell division. (About 50 bases are lost from each telomere every time a normal cell divides.) Mammalian cells in culture will divide only ~ 50X “Telomere theory of aging”—cells senesce and die when the telomeres are gone. Evidence?: Over-expression of telomerase activity extends the life span of cells. Reactivation of Telomerase activity in cancer cells

Telomerase:

Telomerase Ma intains telomere length by restoring telomeres to the 3’-ends of chromosomes A ribonucleoprotein complex Consist of a 126 kDal RNA-dependent DNA polymerase, other proteins and a 450-nt RNA PRESENT IN GERM LINE CELLS AND ABSENT IN SOMATIC CELLS The telomerase polymerase is a “reverse transcriptase” The template sequence from the telomerase RNA is AxCy complementary to TxGy sequences on 3 `end of linear chromosome (where x & y=1to4) Uses the 3’-end of the DNA as a primer and adds successive repeats to it (TTTTGGGG for Oxytricia; TTTAGG for humans ). Ciliated protozoans such Oxytricia and Tetrahymena are good models for studying telomerase activity because they have thousands of “minichromosomes.” Oxytricia has 107 gene-sized chromosomes in its macronucleus.

Protection of telomeres in absence of telomerase:

Protection of telomeres in absence of telomerase In lower eukaryotes(having short telomeres), the overhanging strand is protected by specific binding proteins In higher eukaryotes {incl.mammals},the overhanging single strand forms a specialized structure ,The T loop with the aid of two proteins-TRF1 and TRF2. T loop makes 3` end in accessible to nucleases and other DNA repair enzymes.

FORMATION OF T LOOP:

FORMATION OF T LOOP

What happens to the histones?:

What happens to the histones? Nucleosomes are disrupted during replication Each ds strand gets half New histones added by chromatin assembly factors

Slide 29:

An experiment similar to Meselson and Stahls’ shows that the new octamers are a random mixture of old and new histones.

Summary:

Summary ORC complex assembles at multiple origins ORCs associate with Cdc6 & Cdt1 and are “licensed” MCM/helicase complex is loaded on to the origin Ordered assembly of additional complexes leads to a competent pre -Initiation complex In S-phase, cell cycle regulated kinases trigger replication After initiation the pre-IC complex is dismantled to prevent premature re-replication without cell cycle Origins in most eukaryotes are very poorly defined High conservation of function from Bacteria to humans –though mechanistic details may differ in each species

Slide 31:

THANKS FOR YOUR PATIENCE