ADDITIONAL PROTEINS REQUIRED
sliding-clamp proteins
brace proteins
brace proteins - called the gamma complex in E. coli. These proteins recognize and bind DNA at the junction between the primer and the template, serving as a brace which helps the polymerase remain bound to the template.
sliding-clamp proteins- called the beta protein in E. coli . These proteins bind adjacent to the brace proteins, forming a ring around the template DNA. The ring formed by the sliding clamp maintains the association of the polymerase with its template as replication proceeds.
See figure 5.8 in text.
As the DNA unwinds, there is a tendency for the helix to become to tightly supercoiled. (See Figure 5.10)
If not relieved, eventually this would interfere with the ability of helicase to unwind the helix.
It is the job of the topoisomerases to relax the supercoiling by making either single-stranded breaks in the DNA (Type 1 topoisomerases) or double-stranded breaks in the DNA (Type 2 topoisomerases). The topoisomerases are also needed to unravel the newly replicated circular DNA molecule that has a tendency to become intertwined.
See FIGURE 5.11 for complete model of the E coli replication fork.
PROOF-READING ACTIVITY Both pol I and pol III possess 3' to 5' exonuclease activity. If an incorrect nucleotide is added during replication, the enzymes reverse direction and remove the nucleotides one nucleotide at a time. When incorrect nucleotides have been removed, the enzymes continue polymerization in the 5' to 3' direction.
Pol III
very complex enzyme
In its most active form it is associated with nine other proteins to form the pol III holoenzyme (referring to the fact that the enzyme is composed of several subunits).
Smallest aggregate of the holoenzyme which retains polymerizing activity is referred to as the Core Enzyme.
Pol III shares with pol I a requirement for a template and a primer However, pol III cannot polymerize at a nick. There must be a gap in the sequence .
Pol III does have 3'-5' exonuclease activity - PROOF READING FUNCTION.
NO 5'-3'exonuclease activity.
Enormous size of chromosomes and geometric complexity imposed by nucleosomal structure.
One way of overcoming these problems is to have multiple iniation sites. DNA of Drosophila has about 5000 initiation sites. Each site is separated by ~30,000 bases and each site replicates bi-directionally. In Drosophila egg, # of initiation sites goes up to 50,000 and DNA replicates in only 3 minutes.
Human DNA appears to have ~ 30,000 origins of replication.
Very little known about origins in higher eukaryotic cells.
Eukaryotic cells contain five DNA polymerases: alpha, beta, gamma, delta, and epsilon.
Polymerase gamma is located in the mitochondria and is responsible for replication of mitochondrial DNA. The other four enzymes are located in the nucleus.
Polymerases alpha, delta, and epsilon are most active in dividing cells, suggesting that they function in replication. However, no function for epsilon in DNA replication has yet been discovered [may function in DNA repair because it has been shown to be essential for cell survival.
In contrast, polymerase beta is active in nondividing and dividing cells suggesting that it may function primarily in DNA repair.
Typical animal cell has 20,000 to 60,000 molecules of polymerase alpha , which is one of the two DNA polymerases thought to be involved in replication of nuclear DNA. Polymerase alpha is responsible [in part] for lagging strand synthesis, while polymerase delta synthesizes the leading strand. Polymerase alpha also has primase activity in association with primase in many eukaryotic systems. May just be complexed with the primase in a polymerase a- primase complex. The primers are then extended by polymerase delta.
Brace proteins - termed replication factor C {RFC} in eukaryotes
Sliding Clamp proteins- termed proliferating cell nuclear antigen {PCNA} in eukaryotes.
Although eukaryotic chromosomes are composed of linear rather than circular DNA molecules, their replication also requires topoisomerases: otherwise the complete chromosomes would have to rotate continually during DNA synthesis. Suggestion is that it also untangles newly replicated loops of DNA in the chromosome of eukaryotes.
ALSO
must have a dissociation of DNA and histone octamers - and a histone-DNA reassociation step.
Nuceleosomes apparently form very rapidly following replication of the daughter strand. The synthesis of histones, therefore, must occur simulataneously with DNA replication.
PROOF-READING FUNCTION
3' to 5' exonuclease activity has also been shown to be characteristic of DNA polymerase alpha and delta.
Replicating the Telomeric Ends of Eukaryotic Chromosomes
The extreme 5' ends of the eukaryotic chromosome cannot be replicated by DNA polymerase because of the requirement for 5' to 3' synthesis. Special mechanisms required.
Telomeres consist of tandem repeats of simple-sequence DNA and they are replicated by the telomerase which catalyzes synthesis without a DNA template.
Telomerase is actually a reverse transcriptase - enzymes that synthesize DNA from an RNA template. Telomerase carries its own template RNA, which is complementary tohe telomere repeat sequences. Telomerase, therefore, is able to generate multiple copies of the telomeric repeat sequences.
See figure on page 189 of text.
Mechanism of telomerse action elucidated in 1985 by Carol Greider and Elizabeth Blackburn.