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FM1010 Biochemistry > TMC5 > Flashcards

TMC5 Flashcards

1
Q

What is the difference between G1, G0, S, G2 and M phases of the cell cycle?

A

G0 - Non-dividing state
G1 - Cell grows and prepares for cell division, checks previous cycle was completed correctly and cell cycle checkpoints, cell arrests if checkpoints aren’t met
S - DNA replication phase
G2 - Cell prepares for mitosis by growing, producing proteins, increased organelles
M - mitosis and cell division

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2
Q

What is an origin of replication?

A

The points on DNA where replication begins

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3
Q

What is the difference between NTPs and dNTPs?

A

NTPs - precursors of RNA
dNTPs - precursors of DNA

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4
Q

List the items required by DNA polymerase for DNA synthesis

A

dNTPs
Primers
Template strand

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5
Q

What are the differences between RNA polymerase and DNA polymerase?

A

RNA polymerase:
De novo
Produces a single strand
DNA:
Not de novo
Produces a double strand

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6
Q

What is a primer in DNA synthesis?

A

The start point of DNA synthesis by DNA polymerase, synthesises from the 3’ end of an existing piece of RNA/DNA (primer)

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7
Q

Explain how DNA polymerase synthesises a new strand of DNA.

A

Uses one strand of DNA as a template
Synthesises DNA onto the 3’ end of a primer
Attaches a dNTP onto the 3’ OH group of a deoxyribose (5’ to 3’)
Extends a primer annealed to a template
Catalyses the formation of a phosphodiester bond
High energy dNTP is consumed in the reaction releasing pyrophosphate

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8
Q

What is the direction of DNA synthesis?

A

5’ to 3’

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9
Q

Describe the properties of DNA polymerase.

A

Purified DNA polymerase can replicate DNA in vitro
Error rate is 1 in 100,000
Proofreading carried out by a 3’-5’ exonuclease activity
Needs a primer

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10
Q

What is the basic DNA polymerase error rate?

A

1 in 100,000

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11
Q

Describe proof reading by DNA polymerase.

A

Decreases error rate in DNA synthesis by 100 fold
Carried out by a 3’ to 5’ exonuclease activity in DNA polymerase (digests DNA 3’ and moves toward 5’)
Nucleotide misincorporated during DNA replication
Immediately slows down polymerisation by DNA pol by 10,000 fold
Increases the 3’ to 5’ exonuclease activity

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12
Q

Describe mismatch repair

A

About 1% of the time, a misincorporated nucleotide is not excised by proofreading and remains in DNA creating a mismatch base pair
Corrects mismatched base pair
Mismatch repair system must be able to discriminate between the old strand of DNA and the newly synthesised DNA so that it can remove the mismatched base from the new strand

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13
Q

Explain the differences between DNA polymerase proof reading and mismatch repair.

A

Proofreading by the DNA polymerase during DNA synthesis reduces error by about 100 fold.
Mismatch repair (MMR) repair occurs after DNA synthesis and reduces error by another 100 fold.

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14
Q

What is HNPCC and what are the characteristics of HNPCC?

A

Hereditary Nonpolyposis Colorectal Cancer
Defects in mismatch repair system
Cancer of colon and rectum
Increased risk of cancers of GI tract and endometrium
3 relatives over 2 generations with colorectal cancer, 2 must be first degree relatives, 1 must be under 50, FAP must be excluded

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15
Q

What is the genetic basis of HNPCC?

A

Mutations in any of the mismatch repair genes for the proteins that recognise different forms of mismatched base pairs in DNA cause HNPCC
MLH1, MSH2, MSH6, MSH3, PMS1, PMS2

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16
Q

Explain how DNA replication is initiated.

A

Recognition:
Complex of proteins binds the origin of replication - key one being DnaA
ORC binds to ori in eukaryotes
Melting:
Proteins at ori recruit proteins and melts DNA at ori
Unwinding:
Helicases unwind DNA and cause a replication bubble at ori
Single stranded binding protein binds to the single stranded DNA and keep it single stranded so that replication can proceed
Recruitment:
Proteins assembled at ori must recruit DNA polymerase
Primer synthesis:
RNA primer is synthesised at ori by DNA primase
Supercoiling builds up at edges of opening
Supercoiling relieved by topoisomerases I and II in humans and DNA gyrase in prokaryotes
DNA polymerase then starts synthesising

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17
Q

List the proteins involved in initiating DNA synthesis and each of their functions.

A

DnaA binds at ori
ORC binds at ori in eukaryotes
ORC recruited proteins melt DNA
Helicases unwind DNA and cause replication at bubble
Single stranded binding protein binds to the single stranded DNA and keep it single stranded so that replication can proceed
RNA primer is synthesised by DNA primase
Topoisomerase relieves supercoiling
DNA gyrase relieves supercoiling in prokaryotes
DNA polymerase synthesises DNA

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18
Q

Explain leading and lagging strand DNA synthesis.

A

Leading strand is the DNA synthesised from the RNA primer that can continue all the way to the end of the DNA molecule
Two replication forks one travelling right and the other left
DNA replication occurs on both template strands at the same time in the same direction
5’-3’ action on one of the strands is achieved by Okasaki fragments on one strand being joined up to allow overall progress in a 3’-5’ direction
Okasaki fragments are started from a RNA primer
Sealed by DNA ligase after DNA polymerase removes primers
DNA pol I binds to 3’ of Okaskai fragment and extends fragment by synthesising new DNA til it meets a primer
DNA pol I has 5’ to 3’ exonuclease activity
DNA pol I then dissociates after primer is degraded leaving a nick which is sealed by DNA ligase
In humans, primer is removed by Fen-1 and RNAseH and the gap is closed by DNA polymerase

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19
Q

What is the function of DNA polymerase I in E. Coli?

A

Proofreading, primer removal

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20
Q

What is the function of DNA polymerase III in E. Coli?

A

Lagging strand synthesis
Leading strand synthesis
Proofreading

21
Q

What is the function of DNA helicase in E. Coli?

A

Unwinds DNA from helix

22
Q

What is the function of DNA ligase in E. Coli?

A

Seals nicks in DNA

23
Q

What is the function of DNA primase in E. Coli

A

Primer synthesis

24
Q

What is the function of DNA topoisomerase?

A

Relieves supercoiling in front of replication fork

25
Q

What is the function of DNA gyrase in E. Coli?

A

Relieves supercoiling

26
Q

What unwinds DNA in humans?

A

ORC / DNA helicase

27
Q

What synthesises primers in humans?

A

DNA polymerase alpha / DNA primase

28
Q

What is in charge of leading strand synthesis in humans?

A

DNA polymerase epsilon

29
Q

What is in charge of lagging strand synthesis in humans?

A

DNA polymerase delta

30
Q

What is in charge of proofreading in humans?

A

DNA DELTA / epsilon

31
Q

What is in charge of primer removal in humans?

A

Fen-1
RNAseH

32
Q

What is in charge of primer extension after primer removal in humans?

A

DNA polymerase delta

33
Q

What seals the nicks of DNA in humans?

A

DNA ligase I

34
Q

What can kill the cell which is usually ceased in cancer cells?

A

Supercoiling

35
Q

What is the difference between leading strand and lagging strand synthesis?

A

1.A leading strand is the strand which is synthesized in the 5’-3’direction while a lagging strand is the strand which is synthesized in the 3’-5’ direction.
2.The leading strand is synthesized continuously while a lagging strand is synthesized in fragments which are called Okazaki fragments.
3.Leading strand synthesis does not require an RNA primer while a lagging strand synthesis requires RNA primase.

36
Q

Explain how DNA replication terminates in prokaryotes/E. Coli.

A

The genome is circular
The replication fork proceeds around the circle to a termination site
The termination site is opposite the ori
The protein tus binds to the 10 ter sequences at the termination site
Tus action with other proteins results in completion of replication of the genome
Genomes are intertwined and resolved/separated by topoisomerase IV

37
Q

Explain the difficulty in replicating the ends of linear chromosomes

A

Lagging strand causes problems as DNA polymerase requires a primer with a free 3’ OH group and RNA primers cannot remain in DNA meaning that the chromosome shortens each time its replicated

38
Q

What is the role and structure of telomeres

A

Eukaryotic chromosomes have telomeres that protect the end of a chromosome from damage
Telomeres are composed of repeated TTAGGG sequences
Telomerase maintains the length of telomeres
Telomeres contain TERT and TERC (adds TTAGGG)
The no. of TTAGGG repeats at the 3’ end of the
chromosomes increases with telomerase action
Telomerase extends chromosome by using a sequence on its own RNA as a template
Telomerase then translocates 6 bases

39
Q

Describe the relationship between telomerase and aging and cancer cells.

A

Telomere repeats are lost during every
mitosis as telomerase is repressed in somatic cells - eventually telomere shortening hits a threshold that causes senescence or cell death
Telomerase is active in >85% of cancer cells and telomere lengthening is strongly correlated with cancer.

40
Q

Describe the types of RNA genomes found in viruses.

A

2 types of viruses: Double stranded RNA viruses and retroviruses
Double stranded RNA virus:
encode their own polymerase for replication called RNA dependent RNA polymerase
Retroviruses:
Copied into DNA by viral encoded reverse transcriptase
DNA inserted into genome of host and inserted genome is transcribed by RNA polymerase

41
Q

What is a silent mutation, a missense mutation and a nonsense mutation?

A

Silent - no change in amino acid sequence
Missense - changes the code from one amino acid to another e.g. gcu codes A but changed to ccu coding P
Nonsense - changes the code to a stop codon e.g. aaa codes K but changed to uaa

42
Q

List the different type of agents that inhibits DNA replication

A

Nucleotide triphosphate inhibitors
Antimetabolites
Inhibitors of chain elongation
DNA polymerase inhibitors
DNA damaging drugs
DNA topoisomerase inhibitors

43
Q

What is the effect of methotrexate on DNA replication?

A

Antifolate that inhibits cell division

44
Q

What is the effect of 5-flurouracil on DNA replication?

A

Interferes with pyrimidine production

45
Q

What is the effect of AZT on DNA replication?

A

Causes chain termination as incoming nucleotides cannot be added on to DNA

46
Q

What is the effect of cisplatin on DNA replication?

A

Damages DNA
Common chemotherapy drug

47
Q

What is the effect of etoposide on DNA replication?

A

Inhibits the action of topoisomerase leading to cell death

48
Q

Why is AZT an effective anti-retroviral drug?

A

Reverse transcriptase of HIV has a higher affinity for AZT than dTTP while human DNA polymerases have a higher affinity for dTTP than AZT
AZT can be used selectively against HIV reverse transcription of RNA into cDNA without affecting the DNA polymerase of the host cell

FM1010 Biochemistry (21 decks)

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