Week 4 lecture material Flashcards
What does DNA helicase need to bind to the DNA?
It needs helicase loading protein
What is the role of an indicator protein?
- It binds to the replication origin
- It helps the helicase to bind
- It requires ATP
What is the role of helicase?
Unwind and unzip the dna
think about how many types of helicase exsists?
How does unwinding DNA take place in bacteria?
There are two types of helicase.
But the predominant unwinding takes place in the Lagging strand direction - 5’ to 3’ TEMPLATE
Does helicase need ATP to unwind the Dna?
YES it does
How are the single strands kept seperate?
Single stranded binding protiens help to keep the strands seperate, they prevent H bonding
Which strand is single stranded proteins found on?
Both the leading and lagging strand
What is a primer?
It is a short nucleotide with free 3’ OH end
To begin replication, what does a dna polemerase need?
A bound primer
What is the purpose of the primase in replication?
It is to synthesize an** RNA primer **with a free 3’OH end so dna polemerase can use it
Which direction does Primase proceeds?
reads 3’ to 5’ and synthesises from 5’ to 3’ just like the Dna polemerase. (it adds onto the 3’ end)
Why is an RNA primer attached to DNA?
Its jsut temproray, otherwise the process was thought to be very slow and inaccurate, so dna pol starts off with a primer
What is a primosome?
It is primase + helicase struck together
What is the role of the sliding clamp?
DNA polemerase does not hold onto the dna strand very well, so it needs a sliding clamp to hold it in place.
How are okazaki fragments linked together on the lagging strand?
The repair nucleases remove the okazaki fragments, and then dna polymerase fills in the gap between the other two fragements. Next the nick seeling occurs.
How does Nick seeling exactly happen?
When the DNA polemerase fills in the gap, nick sealing basically forms locall covalent bonds so the DNA filling can be fused together. DNA ligase forms the nick sealing.
What are the gaps in where Okazaki fragments were?
Nick’s
Why does the lagging strand form a loop?
It allows for a replisome
What is Replisome?
It is all the molecular machienes (like DNA polymerase, Helicase etc) working together.
What is the winding problem with DNA?
As DNA helicase starts to unwind from the lagging strand template, the DNA wants to rotate. But there are proteins that want to hold it concrete.
So unzipping it causes steric strain, making the DNA form loops.
How does topoisomer help?
It reduces the tortional stress ahead of the helicase by relieving the stress by free rotation of DNA around the phosphodiester bond opposite the single strand break, and the reseals the strands again.
Do orgainims with all shapes of DNA have the unwinding problem?
No, the ones with circular DNA and long linear ones do (eukaryotes)
What are the problems that occur at the ends on the replicated DNA?
1) Primase is not very good at putting a primer right at the very end.
2) Where it does put a primer, the primer has to be removed
3) After the primer has been removed, if it is a 5’ end, DNA polymerase cannot add nucleotides as it only adds to the 3’ end.
Where does the problem of losing some sequence ends occur?
At the laggin strand ends. This is so cause, when you have leading strands, it goes 3’ to 5’ so DNA polemerase can attach, but lagging start from 5’ end, so it cannot attach and the info is lost.
How does telomerase solve the problem of losing sequence info at the lagging strands?
It adds repetative sequence that is added to the 3’ parential end. (the lagging strand) the repetative sequence is RNA templete in telomerase.
How does telomere replication work?
1) It makes RNA template at the end of the laggin strand (3’ of the parental template lagging)
2) It resembles reverse transcriptase (an rna enzyme)
3) Generates G end
Think of mutations!
What is the problem with relpication in DNA
So sometimes DNA polemerase can make a mistake and put lets say A-G in place of G-C
Now its known that DNA replicates Semi-conservatively, which means when its replicated, AT and GC.
GC is good the mistake is fixed, but in place of A now its gonna be A-T, which is a fixed mutation!
This might produce a bad protein which might hurt us, yet the cell cannot tell what the problem is :(
How is high fidelity maintained in the replication process?
Two mechanisms:
1) 3’ to 5’ exonuclease repair: Removes the incorrect nucleotide and then continues again
2) Strand-directed repair in eukaryotes: If proofreading fails, then this DNA relication repair process occurs.
It is initiated by distortion in the geometry of double helix generated by the mismatch.
How many active sites does DNA polymerase have and what are they?
2 active sites, Editing site, and Polymerizing site
What is the role of the editing active site on DNA polymerase?
When a mismatch occurs, the new complementary strand can go to the editing site where it gets edited and then returns back to polymerizing site.
Ho
How does strand directed mismatch repair work in replication?
So initially as the proof reading falis, MutS comes and binds to the mismatch. it does not know which strand is the replicated one
Then MutL sees MutS and comes to it and they together look for a nick which indicates that it might be a newly complemented strand.
Now MutL cuts and cuts and cuts until it cuts the error. And DNA polymerase comes back and fixes it :)
How do prokaryotes solve the error in replication using the strand direct mismatch?
They work in a similar mechanism, but they look for mythelated adinine instead of Nicks. This is because the new strands do not have the methyl groups.
What factors can cause dna damage?
1) Oxidation
2) Radiation
3) Heat
4) Chemicals
What are pyramidine dimers?
Any two pyramidines can form covalent bonds going up and down between bases
It messes up the spacing.
What are the spontaneous damage that can occur to the DNA?
1) Depurification: This happens when a water molecule hits nucleotide at the wrong place at the wrong time. And seperate the nucleotide and the base
2) Deanimation: C change to U and NH3 is released
What happens if mutaions are not corrected?
It would appear in daughter cells
NOT during replication
What are general ways DNA can be fixed?
Just in general when maybe spontaneous damage occurs
There are 2 ways:
1. Base Excision repair (BER)
2. Nucleotide Excision Repair (NER)
W
How does Base-excirsion repair work to help depurification?
You remove one nucleotide, maybe C, U
How does Nucleotide Excision repair help deamination?
Exclision nuclease comes and cuts a large chunk near the pyramidine dimer, now a helicase comes and removes the part, then the polymerase fixes by adding new base pairs.
What happens in depurification exactly?
Like what bases are created in daughter cells?
G and C’s are mutated into A and T’s
What causes double stranded breaks in DNA strands?
Many forms of radiation
What are the two ways dna double stranded repair is performed?
- Nonhomologous end joining
- Homologous recombination
How does non-homologous end joining work?
Remember it helps to join ends of DNA double strand breaks!
First a nuclease processes the end buy cutting some sequences out
Then ligase joins them.
The problem here is the sequence is lost as ligase just joins both ends.
How does homologous recombination work?
A recombination specific nuclease gets to the break site and then uses the other homologous sequence.
The double stranded break is repaired using the undamaged sequence as the template.
There is no loss in this method.
Out of the 2 mechanisms for fixing double break DNA which one is quick and which is slow?
Non-homologous end joining is quick and dirty
Homologous recombination is slowww but accurate.
