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Year 1 - Biochemistry > 3.1.2 Genes: structure and function (DNA replication) > Flashcards

3.1.2 Genes: structure and function (DNA replication) Flashcards

1
Q

What are the four basic units that make up DNA?

A 
The four nucleotides are:
Adenine (A)
Thymine (T)
Cytosine (C)
Guanine (G)
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2
Q

What is the basic macromolecular structure of DNA?

A

DNA is a double-stranded molecule (with the strands running in antiparallel) that forms a helical structure with major and minor grooves (these are important for the specific interactions of DNA binding proteins like transcription factors)

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3
Q

How do the two strands of DNA hybridise?

A

This is achieved through hydrogen bonds between complementary adenine and thymine bases (x2) and between complementary cytosine and guanine bases (x3)
Due to this bonding pattern, it means that amounts of A and T bases will be the same, as will amounts of C and G bases.

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4
Q

What are the two types of nucleotide?

A

Deoxyribose and ribose sugars are seen in DNA and RNA nucleotides respectively

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5
Q

What is the difference between deoxyribose and ribose sugars? What affect does this have on DNA and RNA?

A

Ribose sugars have an OH group on the 2’ carbon, whereas deoxyribose sugars have a H group.
Both are 5 carbon sugars.
This extra alcohol makes RNA more unstable and also acts as the reactive group for splicing

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6
Q

What is the function of DNA?

A

To store genetic information

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7
Q

What is the function of mRNA?

A

Mediates between nucleic acid and proteins

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8
Q

Which molecule is more stable, DNA or RNA?

A

DNA is far more stable - some RNA molecules have relatively short half lives

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9
Q

Draw out structure of deoxyribose and ribose sugars

A

Do it! Now!

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10
Q

Which bases are purines?

A

Adenine and guanine

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11
Q

Which bases are pyrimidines?

A

Thymine and cytosine (also uracil, replaces thymine in RNA molecules)

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12
Q

How many rings do purines have?

A

2 (shorter word, more rings)

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13
Q

How many rings to pyrimidines have?

A

1 (longer word, fewer rings)

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14
Q

What is a nucleoTide made up of?

A

A base, a sugar and phosphate group(s)

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15
Q

What is a nucleoSide made up of?

A

A base and a sugar

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16
Q

How is nomenclature derived for derivatives of DNA?

A

Fist letter: the base (i.e. C, A, G, T, U)
Second letter: the number of phosphate groups present (m=mono, t=tri etc, the more phosphates present the more negative the molecule is)
Third letter: Always a P, stands for phosphate

Ribonucleotides: RNA
Deoxyribonucleotides: DNA -> these will always have a lower case d in front of the triple letter derivative to signify deoxyribose is present

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17
Q

What bonds are between nucleotides?

A

3’ to 5’ Phosphodiester bonds, formed between an oxygen on the phosphate and an alcohol on the 3’ carbon of the sugar

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18
Q

What is the enzyme that catalyses the formation of phosphodiester bonds in DNA synthesis?

A

Polymerase enzymes (exist for both DNA and RNA)

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19
Q

What are the products of a polymerase reaction?

A

An oligonucleotide/polynucleotide and a pyrophosphate molecule (if one of the species was a triphosphate)

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20
Q

Which end of the DNA is typically phosphorylated?

A

The 5’ end

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21
Q

Which end of the DNA typically contains a free OH group?

A

The 3’ end

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22
Q

In which direction does DNA synthesis/do polymerase enzymes act in?

A

5’ to 3’ (new nucleotides are only ever added to the 3’ end of the molecule)

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23
Q

Why does DNA have directionality?

A

Due to the sugar-phosphate backbone running in a specific direction. Due to the complementary structure of the polymerase enzymes, this allows them to act in only one direction.

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24
Q

The two base pair strands are:

A

Anti-parallel, so run in different directions/have different directionality
Complementary, the nucleotide composition of one strand can be inferred from the nucleotide content of the other strand.

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25
Q

What happens during interphase?

A

Cell replication is completed - the chromosomes are duplicated so that both cells will have a full set of genetic information after cell division in M phase.

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26
Q

How can single stranded nucleic acids be used?

A

As templates - this is a key feature of DNA replication and protein synthesis (transcription)

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27
Q
  • How can it be shown that genes are able to be extracted from cells?
A

Through genetic modifications and the ability to transfer genetic information to cells in vitro

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28
Q
  • What is the physical evidence for DNA structure?
A

This is found through simple treatment of x-ray diffraction through mapping the angle at which X-rays are deflected after interacting with the molecule. This is the method by which Rosalind Franklin derived the structure of DNA.

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29
Q

What is the central dogma of molecular genetics?

A

DNA -> RNA -> Protein (proteins can also affect RNA production, but the process cannot go back to DNA except for in retroviruses)

30
Q
  • Who first proposed the basic structure of DNA?
A

The double helix structure of DNA was first published by Watson and Crick in 1953/1954, but a lot of the work in discovering this was also done by Rosalind Franklin, who was an expert in x-ray crystallography and died before Watson and Crick were awarded their Nobel prize.

31
Q

By what process is DNA replicated?

A

Semi-conservative replication - during replication the two complementary antiparallel strands have to be unwound to act as templates. dNTPs are used to synthesise the new daughter strands, with the necessary bonds being formed by polymerase enzymes

32
Q

Where is the double DNA strand opened for replication?

A

At specific nucleotide sequences known as origins of replication

33
Q

How is the double DNA strand opened for replication?

A

Through the action of DNA helicase enzymes, these break the H bonds between the bases of the two strands

34
Q

Once opened, how are the two strands prevented from re-annealing?

A

Single strand specific binding proteins (SSB), like replication protein A (RPA)
These proteins stabilise an otherwise energetically unfavourable position and prevent strands from re-annealing after the action of DNA helicase.

35
Q

What do DNA polymerase enzymes require to initiate replication?

A

As DNA polymerases are unable to synthesise strands de novo, they require the presence of RNA primers (synthesised by primases), which are short double stranded sequences of RNA.
This is also important for DNA replication in vitro (i.e. PCR)

36
Q

Due to the directionality of DNA, how can both strands/directions be synthesised at once?

A

This is achieved through the presence of leading and lagging strands - on the lagging/unfavourable direction, Okazaki fragments are formed and joined together using ligase enzymes once primers are removed.

37
Q

How is tension created in the strand by the movement of the replication forks overcome?

A

Topoisomerase enzymes relieve or induce supercoiling in the strand - in eukaryotes this is achieved through making a cut in one of the DNA strands and then allowing the two strands to rotate around each other and release the tension.

38
Q

How are the telomeres on the lagging strand replicated?

A

This is achieved through the action of telomerase enzymes

39
Q

How are the origins of replication prepared for DNA synthesis?

A

The double helix is opened with the aid of initiator proteins (large complex), which also prevents the refolding of the strands.
These proteins have been activated via phosphorylation by specific kinases - these in turn were activated during S phase of the cell cycle.

40
Q

How many origins of replication are there on a prokaryotic chromosome?

A

Just one

41
Q

How many origins of replication are there on a eukaryotic chromosome?

A

Multiple (there are a total of 20k in the human genome)

42
Q

Why are there multiple origins in eukaryotic chromosomes?

A

To increase the rate of transcription (pace = ~50nts/s, average length of a chromosome is 150 x 10^6)

43
Q

How are origins of replication activated during S phase?

A

Pre-replication complexes (preRCs, protein complexes) are assembled during G1, and are activated throughout S phase, although not all at the same time - however all will have been activated by the end of S phase.
Bidirectional replication is achieved from activated origins.

44
Q

What is the action of primase/DNA polymerase alpha?

A

This enzyme forms short RNA primers on both strands at replication origins, allowing the action of DNA polymerase.

45
Q

In which direction are new nucleotide added to the strand?

A

5’-3’, so new nucleotides will be added at the 3’ end of the strand being synthesised, so 5’-3’ does NOT refer to the directionality of the template strand.

46
Q

What is the leading strand?

A

This is the stronger/primary direction of synthesis. Synthesis here is normal - the enzyme keeps working until instructed to terminate/the end of another newly synthesised strand is reached.

47
Q

What is the lagging strand?

A

This is the weaker/secondary direction of synthesis due to the location of the sites of origin. Okazaki fragments are formed that need to be joined together using ligase enzymes.

48
Q

Which way do replication forks move?

A

In both directions! Bidirectional.

This increases efficiency.

49
Q

What provides the energy for the action of DNA helicases?

A

The hydrolysis of ATP

50
Q
  • Which DNA polymerase carries out DNA replication?
A

DNA polymerase delta

51
Q
  • What is the function of PNCA?
A

This protein acts as a clamp, tethering the DNA polymerase delta enzyme to the DNA strand and displacing the primase. This allows synthesis to occur in long segments.

52
Q
  • What is the function of RFC?
A

This protein loads the clamp onto the DNA and allows the PNCA to encircle the DNA in an active/ATP consuming process.

53
Q
  • What is the purpose of FEN 1?
A

This protein removes the RNA flap once Okazaki fragments are completed

54
Q
  • What is the function of RNASe H?
A

This protein degrades the removed RNA primer

55
Q

What is the function of DNA ligase 1?

A

This enzyme fuses/ligates adjacent completed Okazaki fragments together.

56
Q

What analogy is used for DNA synthesis?

A

‘Back stitching’, due to the process occurring in both directions

57
Q

What pushes aside the primer of an adjacent Okazaki fragment?

A

The primer is pushed aside by the DNA polymerase delta and DNA helicase complex.

58
Q

What is the function of telomerases?

A

These enzymes extend the telomeres (at the ends of the chromosome)

59
Q

What is the function of a telomere?

A

Telomeres prevent end to end fusions of the chromosomes as they clearly differentiate the END of a chromosome through 100s-1000s of copies of tandem repeats (TTAGGG)
* Protected by a shelterin protein complex

60
Q

Why do lagging ends shorten at the telomeres?

A

This is because the DNA polymerase delta enzyme has to act in a 5’ to 3’ direction, so is only able to move in one direction along the antiparallel strands.
The enzyme can only initiate synthesis at an origin of replication, where primase enzymes are able to synthesise a primer. As there is not one of these origins at the end of the telomere, there is no template for the primase so the action of DNA polymerase delta is also prohibited.
This means that a stretch of DNA is unable to be replicated, causing the code that was able to be synthesised on the leading strand to be removed (as single stranded) and for the telomeres to shorten.

61
Q

What happens when the telomeres become too short?

A

The cell enters replicative senescence, where it will no longer proliferate/leaves the cell cycle. This will also eventually lead to cell death.

62
Q

What type of enzyme is a telomerase and how does it work?

A

A reverse transcriptase (contains a ribonucleo protein), so is able to synthesise a DNA strand that is complementary to an RNA template.
This template is shifted along the parent strand, and the telomerase enzyme extends the DNA of the parent strand, synthesising new tandem repeats and an origin of replication.
In this way, the telomerase allows the DNA primase and polymerase delta to create a new Okazaki fragment from that can then be joined onto the end of the telomere, extending the chromosome.

63
Q
  • What is the involvement of telomerases in cancer?
A

If a mutation occurs and the telomerases become permanently active, the cells will never reach senescence as their telomeres keep being extended. This leads to tumorigenesis/neoplasia as somatic cells no longer leave the cell cycle and become almost immortal, being able to proliferate endlessly.

64
Q

How often do errors occur in 5’-3’ polymerisation?

A

1 in every 10^5 nucleotides

65
Q

How often do errors occur in 3’-5’ exonucleolytic proofreading?

A

1 in every 10^2 nucleotides

66
Q

How often do errors occur in strand-directed mismatch repair?

A

1 in every 10^2 nucleotides

67
Q

What is the combined error of DNA synthesis?

A

Errors occur 1 in 10^9 nucleotides

68
Q

How are mutations that occur during replication dealt with?

A

The polymerase enzyme itself has a proofreading subunit
3’-5’ exonucleolytic proofreading recognises if a base pair isn’t complementary and corrects the DNA polymerase and the chain. Acts in the opposite direction to the polymerase enzyme.

69
Q

What happens if an incorrect nucleotide is added to the newly synthesised strand?

A

It causes a disruption in the sugar phosphate backbone, which will cause polymerase enzymes with 3’-5’ exonuclease activity to shift from polymerase to exo-activity.
The presence of an unpaired 3’ OH group of the incorrect nucleotide blocks synthesis by polymerase enzyme.
The ‘wrong’ nucleotide is removed (needs to be phosphorylated, then is removed from the strand), and then polymerisation is resumed.

70
Q

How is chromatin structure recreated/maintained?

A

This is achieved through the action of histone chaperones, as all histones and nucleosomes have to be removed during DNA replication.
The chaperone proteins catalyse histone removal upstream and replacement downstream of the replication fork, allowing chromatin structure and any epigenetic/histone modifications and methylation of DNA (controlled by other enzymes) to be maintained and replicated on the newly synthesised strand.

71
Q

How many histones are newly synthesised on the daughter strands?

A

50% are inherited, 50% are newly synthesised.

Year 1 - Biochemistry (8 decks)

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