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MCB > MCB3 - DNA Mechanisms: Replication And Repair > Flashcards

MCB3 - DNA Mechanisms: Replication And Repair Flashcards

1
Q

Define DNA replication.

A

Process by which a copy of a DNA molecule is made.

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2
Q

What are the various phases of the cell cycle and give brief overview of each.

A

G1 - prior to DNA synthesis, growth phase 1
G0 - cells which stop dividing
S - DNA replication synthesis phase
G2 - growth phase 2 between DNA replication and mitosis
M - mitosis

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3
Q

Which method is used for DNA replication.

A

Semi conservative, one strand is maintained from the parent DNA molecule, which is used as a template strand to produce a new complementary strand of daughter DNA

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4
Q

Define conservative DNA replication.

A

Original DNA molecule is conserved. New DNA molecule made.

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5
Q

Define dispersive DNA replication.

A

Parent DNA molecule is broken apart and put back together with new DNA sections creating two new DNA molecules with some parent DNA and daughter DNA

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6
Q

Define a primer.

A

Short DNA nucleic acid sequence providing starting point for DNA synthesis. Produced by primase enzymes. Removed

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7
Q

What is DNA helicase and it’s role.

A

Used to break hydrogen bonds between DNA strands using ATP as energy source

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8
Q

How does DNA helicase work on the whole DNA molecule.

A

DNA helicase binds ATP and ADP +Pi in synchronised manner. Conformational changes occur cause the DNA helicase to pull the DNA molecule through its various binding sites.

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9
Q

Discuss the DNA helicase in bacteriophage T7

A

Contains hexagonal arrangement of 6 identical subunits but re not symmetrical. 6 potential binding sites which at any one time have 2 ATP, 2 ADP+Pi and 2 empty. Continuously interchange and pull the DNA molecule through through oscillations.

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10
Q

In which direction does DNA polymerase synthesise DNA.

A

DNA polymerase starts from 3’ end of template strand meaning that it builds the new DNA from a 5’ to 3’ direction.

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11
Q

What three things are required for elongation

A

Template strand. Oligonucleotide primer (acts as seed for growing DNA synthesis). dNTPs supply (deoxynucleotide triphosphates)

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12
Q

Discuss the mechanism of the chemical reaction that occurs during chain elongation.

A

Innermost phosphorus atom of incoming dNTP undergoes nucleophilic attack by the 3’ -OH group on primer or previous nucleotide. Phosphodiester bond is formed. Pyrophosphate released.

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13
Q

What is pyrophosphate.

A

Molecule released from dNTP during chain elongation when phosphodiester bond is formed.

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14
Q

Define replication fork.

A

Point at which DNA replication occurs.

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15
Q

Discuss why leading and lagging strand is present.

A

DNA polymerase can build DNA in 5’ to 3’ direction only meaning that one strand is more difficult to synthesise due to the anti parallel nature of DNA molecules.

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16
Q

What are the names of the fragments in lagging strands.

A

Okazaki fragments.

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17
Q

What mechanism is used to build the lagging strand into a Fully function strand.

A

Back stitching mechanism by piecing together all the Okazaki fragments.

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18
Q

Define the end replication problem.

A

No free 3’ -OH group at end of lagging strand to ensure that the entire chromosome is replicated. Without correction, the chromosome would progressively shorten.

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19
Q

What is a telomere.

A

G rich sequence present at the end of chromosomes. Allows recognition by telomerase enzymes.

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20
Q

How does a telomerase to fill the end of the chromosome during DNA replication, the involvement of DNA polymerase and what type of DNA polymerase.

A

RNA template within telomerase elongates parental strand in 5’ to 3’ direction. DNA polymerase alpha completes lagging stand. Carried DNA primase as a subunit allowing a starting primer to be formed and then removed.

21
Q

Discuss priming of the leading strand in DNA replication.

A

RNA primer attached to the 3’ end of template strand, by DNA primase. Free 3’ -OH group at end of RNA primer allows chain elongation. Growth of DNA strand occurs in 5’ to 3’ direction.

22
Q

Discuss priming of the lagging strand.

A

Multiple RNA primers (produces by DNA primase) exist at various points along the template strand. Chain elongation happens producing Okazaki fragments. Ribonuclease erases RNA primers. DNA polymerase fills gap whilst DNA ligase seals gap.

23
Q

Discuss single strand DNA binding proteins.

A

Prevent H bonds reforming after DNA helicase breaks the bonds to unwind the DNA molecule.

24
Q

Discuss purpose of sliding clamp during DNA replication.

A

Helps to tether DNA polymerase to the DNA strand.

25
Q

Why do DNA mutations occur and discuss general ways of fixing mutations.

A

DNA polymerase is not 100% efficient. Intrinsic proof reading activity occurs which makes up for any reduced efficiency.

26
Q

Define DNA mutations and their effects.

A

Chasing in DNA sequence away from that of the general population. Position on chromosome determines effect of DNA mutation (phenotype)

27
Q

Define DNA variants.

A

Any difference in DNA between a population is referred to as a variant as the normal sequence is commonly unknown.

28
Q

What are the five systems for DNA repair.

A 
Direct repair. 
Mismatch repair. 
Excision repair. 
Non homologous end joining.
Homologous directed repair.
29
Q

Give examples of mistakes that may occur during DNA replication leading to mutations.

A

Mismatched nucleotide pairs. Chemical/physical action of mutagens. Break in DNA caused by clastogens, leading to mutations.

30
Q

Define a clastogen.

A

Mutagenic agents giving rise to disruptions or breakages of DNA.

31
Q

Discuss direct repair as a DNA repair system with example.

A

Converts damaged nucleotides back to their original state. Alkylate guanine can pair with thymine instead of cytosine. MGMT enzyme transfers alkyl group to itself preventing mismatch.

32
Q

Discuss excision repair as a DNA repair system, with example.

A

Works on removal of damaged nucleotide (relatively minor DNA damage) and then filling of gap created. DNA glycosylase excises damaged nucleotide, by flipping the damaged base and cutting beta-N-glycosidic bond between base and sugar. Phosphodiester bond on 5’ side of AP site (baseless site) is cut by AP endonuclease. 3’ side cut by endonuclease or phosphodiesterase. Gap filled by DNA pole RNase and sealed by DNA ligase.

33
Q

What is the role of DNA glycosylase.

A

Removes damage nucleotide during excision repair. Flips the damaged nucleotide and cuts beta-N-glycosidic bond between base and sugar, creating baseless site known as AP site.

34
Q

Define an AP site.

A

Baseless site created after DNA glycosylase removes damaged nucleotide

35
Q

Discuss difference between base and nucleotide excision repair.

A

Base excision repair is one base only. Nucleotide excision repair is multiple bases and includes the entire nucleotide.

36
Q

Discuss mismatch repair and when it is required. Discuss example in e.Coli.

A

Detects absence of complementary base pairing by detecting changes between parent and daughter strand.
Ecoli - MutS protein recognises mismatch. MutH protein binds to daughter strand to allow easy recognition. MutH cuts the DNA strand. DNA helicase II detaches segment of strand required, by breaking hydrogen bonds. Single strand is removed and degraded by exonuclease. DNA polymerase I fills the gap which is sealed by DNA ligase.

37
Q

Discuss NHEJ and how it is caused.

A

Non homologous end joining. Occurs during DNA replication or due to ionising radiation and specific mutagens.

38
Q

How does non homologous end joining work to fix DNA.

A

Uses Ku binding proteins which attach to either side of the break. Have high affinity for one another pulling the strands together.fragments joined together by DNA ligase enzyme.

39
Q

Which method of DNA repair is most prone to errors. Why.

A

NHEJ. Some nucleotides can be lost. Recombination may also occur.

40
Q

Briefly discuss homologous directed repair.

A

Uses homologous sequence as a template which originates from a copy of itself existing after DNA replication.

41
Q

Discuss one cycle in PCR.

A

Heated to 95 to separate DNA strand to break hydrogen bonds.
Lowered to 55 to allow primers to hybridise to DNA.
Heated to 72 to allow heat stable DNA polymerase to amplify DNA fragment.

42
Q

Discuss how PCR can be multiplexed.

A

Using fluorophores to label primers

43
Q

What is the multiplexing of PCR.

A

Allows multiple target DNA sections to be amplified.

44
Q

Discuss Sanger sequencing.

A

Allows sequencing of genome, by using principles of PCR.

45
Q

Discuss process of how Sanger sequencing works.

A

Chemically altered nucleotides used (ddNTPs - dideoxynucleotide triphosphoates) which cause the chain to terminate every time one is incorporated. Each time uses ddNTP for one base and the other three are dNTPs. Each segment is put together to make complete sequence. Gel electrophoresis carried out and fragments read from small to large fragments.

46
Q

What is the purpose for electrophoresis.

A

Separates DNA molecules according to fragment size allowing us to determine the sequence.

47
Q

Discuss NGS

A

Next generation sequencing includes processes of Sanger sequencing with new technological advances.

48
Q

Discuss positives and negatives of personal genomics.

A

Positives - allows people to make informed choices. Gives potential for new medicines.
Negatives - invasion of privacy. Gives information about genetic diseases which may not be able to be cured.

MCB (13 decks)

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