DNA replication in eukaryotes:

• Each eukaryotic chromosome is one linear DNA double helix

• Average ~108 base pairs long

• With a replication rate of 2 kb/minute, replicating one human chromosome would require ~35 days.

• Solution ---> DNA replication initiates at many different sites simultaneously.

Rates are cell specific!

Replication forks visible in Drosophila

-not well characterized-at least 4 subunits (tetrameric)-has proofreading activity-highly processive-PCNA dependent loading-makes leading strand-Pol

ε

makes lagging strand

Eukaryotic replisome

not characterized completely

POLYMERASE SWITCHING

Replicon ModelReplication of circular DNA inE. coli

1. Two replication forks result in a theta-like () structure.

2. As strands separate, positive supercoils form elsewhere in the molecule.

3. Topoisomerases relieve tensions in the supercoils, allowing the DNA to continue to separate.

Models of DNA replication

Rolling circle model of DNA replication:

1. Common in several bacteriophages including

(ds DNA) and Φx174 (ss

circular DNA).

2. Begins with a nick at the origin of replication.

3. 5’ end of the molecule is displaced and acts as primer for DNA synthesis.

4. Can result in a DNA molecule many multiples of the genome length (and make multiple copies quickly).

5. During viral assembly the DNA is cut into individual viral chromosomes.

Termination of DNA replication

FINISHING REPLICATIONCircular chromosomes-

Type II topoisomerase

separate catenanes

•

DNA polymerases can only synthesize DNA only in the 5’

to 3’

direction and cannot initiate DNA synthesis

•

These two features pose a problem at the 3’

end of linear chromosomes

Problem at ends of eukaryotic linear ChromosomesProblem at ends of eukaryotic linear Chromosomes

End replication problem

DNA polymerase/ligase

cannot fill gap at end of chromosome after RNA primer is removed. If this gap is not filled, chromosomeswould become shorter each round of replication!

•

If this problem is not solved–

The linear chromosome becomes progressively shorter with each round of DNA replication

•

The cell solves this problem by adding DNA sequences to the ends of chromosome: telomerestelomeres–

Small repeated sequences (100-1000’s)

–

TTAGGG repeat in humans

•

Catalyzed by the enzyme telomerasetelomerase•

Telomerase contains protein and RNA–

The RNA functions as the template

–

complementary to the DNA sequence found in the telomeric

repeat

•

This allows the telomerase to bind to the 3’

overhang

Model for telomere replication by telomeraseModel for telomere replication by telomerase

Step 1 = Binding

Step 3 = Translocation

The binding- polymerization-

translocation cycle can occur many times

This greatly lengthens one of the strands

The complementarystrand is made by primase, DNA polymerase and ligase

RNA primer

Step 2 = Polymerization

Telomerase Structure

Reverse transcriptase with RNA template to bind to DNA strands

Regulation for DNA replicationIn Bacteria, hemimethylated origins are resistant to initiation,

delayed methylation leads to delayed initiation at the second phaseDam methylase

REGULATION OF REPLICATIONE. coli-

DnaA•ATP

levels-

SeqA-

binds to hemimethylated

GATC sequences and inhibits methylation

at A by Dam methyl transferase

DNA replication in eukaryotes:

Copying each eukaryotic chromosome during the S phase of the cell cycle presents some challenges:

Major checkpoints in the system

1. Cells must be large enough, and the environment favorable.

2. Cell will not enter the mitotic phase unless all the DNA has replicated.

3. Chromosomes also must be attached to the mitotic spindle for mitosis to complete.

4. Checkpoints in the system include proteins called cyclins and enzymes called cyclin-dependent kinases (Cdks).

REGULATION OF REPLICATION

Eukaryotes: Formation of the pre-replication complex (pre-RC)Pre-RC formation in G1-

composed of four proteins which assemble in an ordered fashion atthe replicator-

ORC, the initiator, recognizes the replicator-

helicase

loading proteins, Cdc6 and Cdt1-

putative replicator fork helicase, Mcm

2-7 complex

Origins of replication are strictly controlled so that they “fire” only once per cell cycle

Errors lead to over replication of specific chromosomal regions.(= gene amplification)

This seen commonly in cancer cells and can be an importantprognostic indicator.

Errors of DNA Replication and Disease

The rate of misincorporation of bases by DNA polymerase isextremely low, however repeated sequences can cause problems.

In particular, trinucleotide repeats cause difficulties which can lead to expansion of these sequences.

Depending where the repeat is located expansion of the sequencecan have severe effects on the expression of a gene or thefunction of a protein.

Several mechanisms for the expansion of trinucleotide repeatshave been proposed, but the precise mechanism is unknown.

From Stryer: Looping out of repeats before replication.

Several inherited diseases are associated with expansion of trinucleotide repeat sequences.

Very different disorders, but they share the characteristic of becoming more severe in succeeding generations due to progressiveexpansion of the repeats