Week 4 lecture material

Question 1

What does DNA helicase need to bind to the DNA?

Answer 1

It needs helicase loading protein

Question 2

What is the role of an indicator protein?

Answer 2

1. It binds to the replication origin
2. It helps the helicase to bind
3. It requires ATP

Question 3

What is the role of helicase?

Answer 3

Unwind and unzip the dna

Question 4

think about how many types of helicase exsists?

How does unwinding DNA take place in bacteria?

Answer 4

There are two types of helicase.
But the predominant unwinding takes place in the Lagging strand direction - 5’ to 3’ TEMPLATE

Question 5

Does helicase need ATP to unwind the Dna?

Answer 5

YES it does

Question 6

How are the single strands kept seperate?

Answer 6

Single stranded binding protiens help to keep the strands seperate, they prevent H bonding

Question 7

Which strand is single stranded proteins found on?

Answer 7

Both the leading and lagging strand

Question 8

What is a primer?

Answer 8

It is a short nucleotide with free 3’ OH end

Question 9

To begin replication, what does a dna polemerase need?

Answer 9

A bound primer

Question 10

What is the purpose of the primase in replication?

Answer 10

It is to synthesize an** RNA primer **with a free 3’OH end so dna polemerase can use it

Question 11

Which direction does Primase proceeds?

Answer 11

reads 3’ to 5’ and synthesises from 5’ to 3’ just like the Dna polemerase. (it adds onto the 3’ end)

Question 12

Why is an RNA primer attached to DNA?

Answer 12

Its jsut temproray, otherwise the process was thought to be very slow and inaccurate, so dna pol starts off with a primer

Question 13

What is a primosome?

Answer 13

It is primase + helicase struck together

Question 14

What is the role of the sliding clamp?

Answer 14

DNA polemerase does not hold onto the dna strand very well, so it needs a sliding clamp to hold it in place.

Question 15

How are okazaki fragments linked together on the lagging strand?

Answer 15

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.

Question 16

How does Nick seeling exactly happen?

Answer 16

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.

Question 17

What are the gaps in where Okazaki fragments were?

Answer 17

Nick’s

Question 18

Why does the lagging strand form a loop?

Answer 18

It allows for a replisome

Question 19

What is Replisome?

Answer 19

It is all the molecular machienes (like DNA polymerase, Helicase etc) working together.

Question 20

What is the winding problem with DNA?

Answer 20

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.

Question 21

How does topoisomer help?

Answer 21

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.

Question 22

Do orgainims with all shapes of DNA have the unwinding problem?

Answer 22

No, the ones with circular DNA and long linear ones do (eukaryotes)

Question 23

What are the problems that occur at the ends on the replicated DNA?

Answer 23

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.

Question 24

Where does the problem of losing some sequence ends occur?

Answer 24

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.

Question 25

How does telomerase solve the problem of losing sequence info at the lagging strands?

Answer 25

It adds repetative sequence that is added to the 3' parential end. (the lagging strand) the repetative sequence is RNA templete in telomerase.

Question 26

How does telomere replication work?

Answer 26

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

Question 27

# Think of mutations! What is the problem with relpication in DNA

Answer 27

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 :(

Question 28

How is high fidelity maintained in the replication process?

Answer 28

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.

Question 29

How many active sites does DNA polymerase have and what are they?

Answer 29

2 active sites, Editing site, and Polymerizing site

Question 30

What is the role of the editing active site on DNA polymerase?

Answer 30

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.

Question 31

# Ho How does strand directed mismatch repair work in replication?

Answer 31

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 :)

Question 32

How do prokaryotes solve the error in replication using the strand direct mismatch?

Answer 32

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.

Question 33

What factors can cause dna damage?

Answer 33

1) Oxidation 2) Radiation 3) Heat 4) Chemicals

Question 34

What are pyramidine dimers?

Answer 34

Any two pyramidines can form covalent bonds going up and down between bases It messes up the spacing.

Question 35

What are the spontaneous damage that can occur to the DNA?

Answer 35

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

Question 36

What happens if mutaions are not corrected?

Answer 36

It would appear in daughter cells

Question 37

# NOT during replication What are general ways DNA can be fixed? | Just in general when maybe spontaneous damage occurs

Answer 37

There are 2 ways: 1. Base Excision repair (BER) 2. Nucleotide Excision Repair (NER)

Question 38

# W How does Base-excirsion repair work to help depurification?

Answer 38

You remove one nucleotide, maybe C, U

Question 39

How does Nucleotide Excision repair help deamination?

Answer 39

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.

Question 40

What happens in depurification exactly? | Like what bases are created in daughter cells?

Answer 40

G and C's are mutated into A and T's

Question 41

What causes double stranded breaks in DNA strands?

Answer 41

Many forms of radiation

Question 42

What are the two ways dna double stranded repair is performed?

Answer 42

1. Nonhomologous end joining 2. Homologous recombination

Question 43

How does non-homologous end joining work? | Remember it helps to join ends of DNA double strand breaks!

Answer 43

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.

Question 44

How does homologous recombination work?

Answer 44

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.

Question 45

Out of the 2 mechanisms for fixing double break DNA which one is quick and which is slow?

Answer 45

Non-homologous end joining is quick and dirty Homologous recombination is slowww but accurate.