MCB3 - DNA Mechanisms: Replication And Repair

Question 1

Define DNA replication.

Answer 1

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

Question 2

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

Answer 2

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

Question 3

Which method is used for DNA replication.

Answer 3

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

Question 4

Define conservative DNA replication.

Answer 4

Original DNA molecule is conserved. New DNA molecule made.

Question 5

Define dispersive DNA replication.

Answer 5

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

Question 6

Define a primer.

Answer 6

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

Question 7

What is DNA helicase and it’s role.

Answer 7

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

Question 8

How does DNA helicase work on the whole DNA molecule.

Answer 8

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.

Question 9

Discuss the DNA helicase in bacteriophage T7

Answer 9

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.

Question 10

In which direction does DNA polymerase synthesise DNA.

Answer 10

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

Question 11

What three things are required for elongation

Answer 11

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

Question 12

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

Answer 12

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.

Question 13

What is pyrophosphate.

Answer 13

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

Question 14

Define replication fork.

Answer 14

Point at which DNA replication occurs.

Question 15

Discuss why leading and lagging strand is present.

Answer 15

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.

Question 16

What are the names of the fragments in lagging strands.

Answer 16

Okazaki fragments.

Question 17

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

Answer 17

Back stitching mechanism by piecing together all the Okazaki fragments.

Question 18

Define the end replication problem.

Answer 18

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.

Question 19

What is a telomere.

Answer 19

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

Question 20

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.

Answer 20

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.

Question 21

Discuss priming of the leading strand in DNA replication.

Answer 21

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.

Question 22

Discuss priming of the lagging strand.

Answer 22

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.

Question 23

Discuss single strand DNA binding proteins.

Answer 23

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

Question 24

Discuss purpose of sliding clamp during DNA replication.

Answer 24

Helps to tether DNA polymerase to the DNA strand.

Question 25

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

Answer 25

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

Question 26

Define DNA mutations and their effects.

Answer 26

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

Question 27

Define DNA variants.

Answer 27

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

Question 28

What are the five systems for DNA repair.

Answer 28

``` Direct repair. Mismatch repair. Excision repair. Non homologous end joining. Homologous directed repair. ```

Question 29

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

Answer 29

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

Question 30

Define a clastogen.

Answer 30

Mutagenic agents giving rise to disruptions or breakages of DNA.

Question 31

Discuss direct repair as a DNA repair system with example.

Answer 31

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.

Question 32

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

Answer 32

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.

Question 33

What is the role of DNA glycosylase.

Answer 33

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.

Question 34

Define an AP site.

Answer 34

Baseless site created after DNA glycosylase removes damaged nucleotide

Question 35

Discuss difference between base and nucleotide excision repair.

Answer 35

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

Question 36

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

Answer 36

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.

Question 37

Discuss NHEJ and how it is caused.

Answer 37

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

Question 38

How does non homologous end joining work to fix DNA.

Answer 38

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.

Question 39

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

Answer 39

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

Question 40

Briefly discuss homologous directed repair.

Answer 40

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

Question 41

Discuss one cycle in PCR.

Answer 41

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.

Question 42

Discuss how PCR can be multiplexed.

Answer 42

Using fluorophores to label primers

Question 43

What is the multiplexing of PCR.

Answer 43

Allows multiple target DNA sections to be amplified.

Question 44

Discuss Sanger sequencing.

Answer 44

Allows sequencing of genome, by using principles of PCR.

Question 45

Discuss process of how Sanger sequencing works.

Answer 45

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.

Question 46

What is the purpose for electrophoresis.

Answer 46

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

Question 47

Discuss NGS

Answer 47

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

Question 48

Discuss positives and negatives of personal genomics.

Answer 48

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.