DNA Replication Flashcards
In what Phase of the cell cycle does DNA replication happen
S phase
How many replication forks do E.coli have and in what direction do they move?
Two that move in opposite (bidirectional) directions
What does helicase do?
Unwinds and separate the 2 DNA strands by breaking the weak hydrogen bonds
What do single-strand binding proteins do?
Attach to keep the two strands of DNA separate and untwisted or stabilize the single strand of DNA
What does topoisomerase do?
Attaches at the 2 replication forks to relieve stress on the DNA molecule as it separates
What does Primase do?
Synthesizes RNA primers to start the addition of new DNA
Where can DNA pol add nucleotides to?
the 3’ end of the template DNA
What does ligase do?
Joins the okazaki fragments to make one strand
In prokaryotes which DNA pol carries out most synthesis and which completes lagging strand synthesis?
DNA pol III carries out most but pol I completes lagging strand synthesis
What is the 3’ exonuclease activity of DNA pol I?
Serves as the proofreading function
What is the 5’ exonuclease activity of DNA pol I?
Functions in both replication and DNA repair
What is the purpose of Mg2+ in replication?
Help the Negative 3’ OH group and alpha-phosphate form the phosphodiester bond by shielding the negative charges and stabilizing the transition states
Do RNA polymerases need a primer to begin synthesis?
No
Which is the leading strand and the lagging strand?
Leading strand goes 5’ to 3’ in the direction of the the opening of the fork. Lagging strand 5’ to 3’ moving away from the opening of the fork.
What does the lagging strand have to do so that it can synthesize the daughter strand in the right direction?
It needs to loop around so the 2 polymerases can go in the same direction.
What is the holoenzyme made up of?
2 DNA Pol III’s, 2 Tau subunits and structural subunits.
What is the role of the sliding clamp?
The sliding clamp is the component of the holoenzyme complex that encircles the DNA and keeps Pol associated with the template strand. It also increases the speed that DNA pol can add bases.
How many types of sliding clamps are there?
There are 3. prokaryotes = beta w/ 2 subunits. Humans = PCNA 3 subunits. Bacteriophage have gp45 3 subunits
Single stranded binding proteins recruit each other in what kind of manner
Cooperative manner
What direction does helicase work in? What is its purpose?
5’ to 3’. Unwind DNA for replication
What is the mechanism for Type 1 topoisomerase?
Topos unwind DNA for replication. Type 1 cuts one strand and unwinds by transferring the phosphodiester bond between the enzyme and the DNA molecule. They do not use energy in this process.
What is the mechanism for Type 2 topoisomerase?
Type 2 cuts both strands of DNA and introduce supercoils. In prokaryotes Gyrase uses ATP energy to cut both strands to switching the positive supercoiling to negative supercoiling to better facilitate unwinding
DNA twisted in the opposite direction of the double helix
Negative supercoiling. Opposite of the natural, right-handed turn of DNA.
DNA twisted in the same direction of the double helix
Positive supercoiling. This is the natural way DNA turns.
What enzyme mechanism is needed in eukaryotes to deal with supercoiling?
(Not gyrase, a different topo II) They only need to relieve positive supercoils downstream of the replication fork because eukaryotic DNA is net negatively supercoiled around histones. Negative supercoils will not be introduced.
In prokaryotes, how many DNA Pol III molecules are replicating the DNA?
3 - 1 on the leading strand and 2 on the lagging strand
How do you initiate replication in E.coli (prokaryote). Then what happens?
The initiator protein binds to the oriC sequence of the chromosome. The initiator binding recruits helicase, primase and pol III complex.
Where do eukaryotes initiate replication?
Sites that have an AT-rich sequence, because it only has 2 H-bonds as opposed to GC region with 3 H-bonds
What enzyme removes RNA primers and adding DNA bases?
DNA pol I
What does DNA pol III need to be able to add a base?
3’ hydroxyl group
What cells have active telomerase?
Germline cells, eggs and sperm. Somatic cells do not have active telomerase.
What is telomerase?
It is an RNA dependent DNA polymerase. Uses RNA as a template but brings in DNA bases AKA reverse transcription
After telomerase extends the 3’ overhang what happens next?
Primase lays an RNA primer comes in and binds to the region and then DNA pol III adds new bases. Then DNA pol I removes the RNA primer.
What do Telomere binding proteins do?
They protect the DNA as well as regulate telomere length.
What is the difference between dNTPs and ddNTPs?
dNTPs have a 3’ hydroxyl group and ddNTPs have a 3’ hydrogen group
How does sanger sequencing work and how does high throughput sequencing work?
Sanger was slower and needed multiple tubes for reactions and was radioactive. High-throughput uses a single tube to carry out all reactions and use fluorescent dye
