Lecture 3 Flashcards
What are DNA polymerases?
Enzymes that catalyze synthesis of DNA polynucleotide chains
What are the general properties of DNA nucleotides?
- Depend on single stranded template DNA
* Can add free nucleotides only to 3’ OH end of an existing polynucleotide
What direction can DNA polymerases work?
In the 3’ to 5’ direction of the leading (already there) strand continuously. In the 3’ to 5’ strand of lagging strand to produce fragments
What is needed before DNA polymerase can work?
An RNA primer called primase
In what direction is the daughter strand made?
In the 5’ to 3’ direction
What are the 3 DNA polymerases of E.coli?
DNA Polymerase I, II, III
What does DNA polymerase III do?
Synthesizes most of the DNA during DNA replication
What does DNA polymerase I do?
Synthesizes some of the DNA during DNA replication. Removes and replaces primers during DNA replication
What does DNA Polymerase II do?
Not required for replication or growth, functions in DNA repair
How are Nucleotides added on to DNA?
Through hydrolysis
What is bound to the nucleotide before it joins the DNA?
3 Phosphates
How are the 3 phosphates used in DNA replication?
Through hydrolysis they are cleaved off phosphates to use the energy to drive the replication machine to keep replicating
What is Primase?
Primase is an RNA polymerase that creates short sequences of RNA called primers
What do primers provide?
A 3’ -OH end for DNA polymerase during replication
What removes RNA primers?
DNA polymerase
What happens to the RNA primer?
It is cleaved by endonuclease
What proofreads for the DNA polymerase?
Polymerase III
What is proofreading known as?
3’ to 5’ exonuclease activity
What does Helicase do?
Unwinds DNA, bind one strand and use ATP
What direction does Helicase work in?
the 5’ to 3’ direction of the strand it is bound to
What do SSB’s do?
Coat the individual strands of DNA and prevent them from re binding to each other
What encodes primase in E.coli?
dnaG. It aslo encodes RNA polymerase
What are the 3 major modes of DNA rplicastion?
Theta Replication
Rolling Circle
Replication of linear DNA in eukaryotes
Where does Theta replication occur?
Bacteria
Where does Rolling Circle replication occur?
Viruses, phages, plasmids, and episomes
What organism does Replication of Linear DNA. occur?
Eukaryotes e.g. humans
What is the structure of replication RNA viruses?
Many are double or single stranded RNA
What causes high mutation rate in viruses?
The fact that they encode their own replication factors that lack proofreading
What is the process of Theta replication?
Because DNA is double stranded and circular DNA unwinds at the replication origin, producing templates for the synthesis of new DNA. A replication bubble forms and this proceeds around the circle. Eventually two new molecules are produced
How does Rolling Circle replication work?
It is initiated by a break in one of the circular strands. And then the inner strand is used as a template from the 3’ end. Cleavage releases the single stranded linear DNA. The linear DNA serves as a template for a complementary strand
What slows down replication in humans?
Nucleosomes and other chromosome proteins
Why do eukaryotes have multiple origins of replication?
To speed up replication of DNA
What is RNA Primer removed by on the lagging strand?
Exonuclease activity of DNA polymerase I and is replaced by the same enzyme
What seals the discontinuous fragments into a single strand?
DNA ligase
What carries out the replication on the leading strand?
DNA polymerase III
What carries out the replication on the lagging strand?
DNA polymerase I
How many origins of replication in Bacteria?
One
How many replication origins do prokaryotes have?
Many, that are long and not well defined
What ends replication for circular DNA?
Specific proteins building to replication termini called ter
What is DNAa?
The protein that activates the initiation of DNA replication in bacteria by being the region that pulls DNA to unwind it
What does Gyrase do?
In DNA replication it removes torsional stress (supercoiling) by creating double strand breaks
In what direction does Helicase work in?
The 5’ to 3’ direction
What does β-clamp do in DNA replication?
Keeps DNA Polymerase III attached to DNA
What type of overhangs do eukaryotic chromosomes have?
3’ overhangs on either end
What do Telomeres do?
Stabilize the ends of a chromosome
How do Telomeres work?
They seal the chromosome end with a protein complex, protecting it’s single stranded end
What is the WRN protein?
A protein that is critical for telomere maintenance and when impaired, causes Werner Syndrome
What do Telomeres contain?
Long simple repeat sequences
What synthesizes Telomeres?
Telomerase
How does Telomerase work?
It uses RNA to extend the 3’ overhang of DNA which gives room for primase to come in and insert RNA which then allows polymerase to fill in the rest of the DNA
Where is Telomerase active?
In germ cells
What does Telomerase need to work?
A 3’ -OH end to synthesize DNA. This is provided by the 3’ overhang
