Genetics Ch. 12.3 (UNIT 3)
What makes up DNA polymerase III?
SLIDES
Five basic components of an active replication fork?
1) Helicase to unwind DNA 2) SSBs to protect strand and prevent secondary structures 3) Topoisomerase gyrase to remove strain ahead of fork 4) Primase to synthesize primers with a 3'-OH group at beginning of each DNA fragment 5) DNA polymerase to synthesize each strand
Characteristics of all E. coli DNA polymerases
1) synthesize any sequence on template 2) synthesize in 5'-3' direction by adding nucleotides to the 5'-OH group 3) uses dNTP's to syntheize 4) require primer for initiation 5) catalyze phosphodiester bond formation by joining 5' phosphate group of incoming nucleotide to the 3'-OH of the preceding nucleotide, cleaving off 2 phosphates in process 6) Produce new strands that are complimentary and antiparrallel 7) Associated with a number of other proteins.
What is DNA polymerase III
A large multiprotein complex that acts as the main workhorse of replication.
What happens after polymerase I has replaced the last nucleotide of the RNA primer with a DNA nucleotide?
A nick remains in the sugar-phosphate backbone of the new DNA strand
What do all DNA polymerases require?
A nucleotide with a 3'-OH group to which a new nucleotide can be added. Thus, DNA polymerase cannot initiate DNA synthesis on a bare template; requires a primer
What is DNA gyrase?
A topoisomerase. Controls the supercoiling of DNA. in replication, it reduces the torsional strain (torque) that builds up ahead of the replication fork as a result of unwinding.
What is DNA helicase and what is its purpose?
An enzyme that breaks the hydrogen bonds existing between the bases of the two nucleotide strands of a DNA molecule. Helicase binds to each replication fork and moves in the 5'-3' direction along the strand, also moving the replication fork. (helicase cannot initiate unwinding; initiator protein must)
What is DNA Ligase, and what is its function?
An enzyme that seals the nicks. Catalyzes the formation of a phosphodiester bond without adding another nucleotide to the strand.
What is primase?
An enzyme that synthesizes short stretches of nucleotides, or primers, to get DNA replication. Synthesizes a short stretch of RNA nucleotides which provides a 3'OH group to which DNA polymerase cna attach DNA nucleotides.
What is DNA polymerase I?
An enzyme with 5'-3' polymerase and 3'-5' exonuclease activities. Also, unlike DNA polymerase III, has a 5'-3' exonuclease activity
How does DNA polymerase III synthesize nucleotide strands?
By adding new nucleotides to the 3' end of a growing DNA molecule. DNA polymerase III adds DNA nucleotides to the primer(s), synthesizing the DNA of both the leading and the lagging strands.
Info on initiation..
Circular bacterial chromosome (E. coli for example) has a single replication origin (otic). Initiator protein binds to oriC and causes a short section of DNA to unwind. Unwinding allows helicase and other single-strand-binding proteins to attach to the polynucleotide strand.
Enzymes proteins active in unwinding
DNA helicase SSBs DNA gyrase (topoisomerase)
What do DNA polymerase's two functions allow it to do?
Efficiently and accurately synthesize new DNA molecules.
How does DNA polymerase III's exonuclease activity work?
If a nucleotide having an incorrect base is inserted into the growing DNA molecule, DNA polymerase backs up and removes the incorrect nucleotide. IT then resumes normal function.
How is replcaiton terminated?
In some DNA molecules, replication is terminated whenever two replication forks meet. In others, specific termination sequences block further replication.
What are the four stages of bacterial DNA replication?
Initiation, unwinding, elongation and termination
Components required for replication in bacterial cells
Initiator protein DNA helicase Single-strand-binding proteins DNA gyrase DNA primase DNA polymerase III DNA polymerase I DNA ligase
Enzymes/proteins active in initiation?
Initiator protein helicase binding?
What are the two enzymatic activities of DNA polymerase III?
Its 5'-3' polymerase activity allows it to add new nucleotides in the 5'-3' direction. Its 3'-5' exonuclease activity that allows it to remove nucleotides in the 3'-5' direction, enabling it to correct errors.
What does it mean to say that DNA polymerase is highly processive?
Means that it is capable of adding many nucleotides to the growing DNA strand without releasing the template: usually holds the template and continues synthesizing DNA until the template has been completely replicated.
What ensures the high processivity of DNA polymerase III?
One of the polypeptide subunits that constitutes the enzyme. Serves as a sliding clamp for the enzyme; encircles the DNA and keeps DNA polymerase attached to the template strand during replication.
How does primase interact?
Primase forms a complex with helicase at the replication fork and moves along the template of the lagging strand.
How are errors minimized?
Proofreading of polymerase III Mismatch repair after replication
What are single-strand-binding proteins (SSBs)
Proteins that attach tightly to the exposed single-stranded DNA right after DNA has been unwound by helicase. Proteins protect the single strand and prevent the formation of secondary structures.
How does elongation occur at the replication fork?
Two polymerase III units, connected to the main unit, synthesize both strands simultaneously. The lagging-strand loops around so that it is in position for 5'-3' replication. Allows DNA polymerase III complex to synthesize both strands simultaneously even though they run in opposite direction.
What does DNA polymerase I do?
Uses its 5'-3' exonuclease activity to remove primers and synthesize a DNA strand in their place in the 5'-3' direction. The removal and replacement of primers appear to constitute the main function of DNA polymerase I. Lower processivity than DNA polymerase III
Primers on leading and lagging strand..
leading strand requires one primer. Lagging strand requires multiple primers at the beginning of each Okazaki fragment. .