Genetic variation in the human genome

Question 1

What is a mutation ?

Answer 1

The process by which a gene undergoes a structural change

Question 2

Explain Point mutations ?

Answer 2

Consist of the change of a single nucleotide in

the DNA sequence. They are divided into transitions (Pu to Pu, Py to Py) and transversions (Pu to Py, Py to Pu)

Question 3

How can mutation occur ?

Answer 3

They can spontaneous (due to errors in genome duplication) or induced (by mutagens)

Question 4

How does DNA polymerase improve fidelity of DNA replication?

Answer 4

By selecting the correct nucleotide that pairs with the templates, and then by proofreading the incorporated nucleotide with 3’ to 5’ exonuclease activity (1 in 107 error rate in E. coli)

Question 5

Further checks on disincorporated nucleotides are then applied by?

Answer 5

The DNA repair pathways

Question 6

Regardless of the fidelity of the DNA polymerase some mis-incorporation of nucleotides is
unavoidable due to ?

Answer 6

Tautomeric shifts of the bases

Question 7

What are Tautomers ?

Answer 7

Structural isomers in
dynamic equilibrium: for DNA bases this is between the keto and enol forms, and amino and
imino forms, and while the former are the more stable ones the latter do occur and can cause
mutation if present at the time of passage of the replication fork

Question 8

In the presence of DNA repeats the DNA polymerases can slip during replication and this
can lead to ?

Answer 8

Insertion or deletion of repeat units

Question 9

Insertions and deletions a frameshift mutation can occur, which change ?

Answer 9

The translational reading frame of a gene

Question 10

Explain a nucleotide repeat expansion disease?

Answer 10

• Huntington disease is an example of these disorders. The Huntington gene has a stretch of less than about 30 CAG triplet repeats (coding for glutamine). If this stretch is expanded to greater than about 40, disease occurs. The expansion might be due to fork slippage, but possibly during repair rather than during replication.

Question 11

DNA is a stable molecule but it is nevertheless subject to damage by a number of agents, including endogenous ones, such as?

Answer 11

• water (hydrolysis)
• compounds that are generated within cells (reactive oxygen species, ROS, such as O2- , H2O2, *OH; oxidative damage)
• and methylation (methyltransferases, S-adenosylmethionine)

Question 12

What can oxidative damage lead to ?

Answer 12

Breakage of the DNA strand backbone. Another
frequent type of damage is attach of bases, especially purines. breakage of the DNA strand backbone. Another frequent type of damage is attach of bases, especially purines.

Question 13

Oxidation of guanine can lead to?

Answer 13

Hoogsteen-type base pairing of the modified base to adenine, resulting in mutation

Question 14

What can hydrolysis of DNA lead to ?

Answer 14

Hydrolysis can for example led to cleavage of the glycosidic bond with loss of a base from the DNA (base deletion: depurinaton or depyrimidination)

Question 15

What is another thing hydrolysis of DNA lead to?

Answer 15

Hydrolysis can also lead to deamination. In the case of cytosine, this can lead to appearance of uracil in DNA, which is normally restricted to RNA. A specific enzyme removes U from DNA (Base Excision Repair pathway)

Question 16

What do methyltransferases?

Answer 16

Add methyl groups to cytosines

Question 17

What are some mutagens that are a source of induced mutations ?

Answer 17

• UV light
• Ionising radiation
• Chemicals

Question 18

What are some physical mutagens include ?

Answer 18

UV and ionising radiation, and heat

Question 19

What are some chemical mutagens and explain ?

Answer 19

1. Base analogs such as 5-
bromouracil (5-Br-dU) have
a stronger propensity to
adopt the tautomeric form
2. Alkylating agents
(ethylmethane sulfonate, EMS) can lead to changes in base pairing properties of bases, thus leading to point mutations
3. Deaminating agents (nitrous acid), also can change the base pairing properties of bases. For example deamination of adenine gives hypoxanthine, which pairs with C (leading to T to C
transitions)
4. Intercalating agents
(ethidium bromide)
usually lead to insertion
mutations, as they change
the spacing between bases

Question 20

Explain the physical mutagens ?

Answer 20

1. UV radiation (at 260 nm) - induces dimerisation of adjacent pyrimidines, especially Ts, producing cyclobutyl dimers: the resulting mutation is generally deletion after replication. Another possible UV product is the (6-4) lesion, linking carbons 6 and 4 of adjacent pyrimidines
2. Heat - stimulate hydrolysis of the glycosidic bond, leading to AP (apurinic/apyrimidinic) sites. About 18,000 AP sites are generated in each human cell
   per day! So repairing them is important
3. Ionising radiation has a complex effect depending on type and intensity. It can lead to full breakage of the double strand

Question 21

About how many purines are lost in each cell every day at 37C in the human body ?

Answer 21

About 18,000 purines

Question 22

About how many deamination events of C to U occur in each human cell per day ?

Answer 22

About 100-500

Question 23

The essential metabolite S-adenosylmethionine acts as?

Answer 23

Weak alkylating agent and methylates position 3 of adenine about 1,200 times per human cell per day

Question 24

Defects in DNA repair can cause human syndromes, which often display the following disease
features:

Answer 24

• Cancer susceptibility (C). Increased mutation
  rate and genome instability is the basis for
  increased cancer rate
• Progeria (P). Some disorders have
  characteristics of accelerated ageing. DNA
  damage leads to cell death and loss, including
  telomere defects
• Neurological features (N). Neuronal death and
  neurodegeneration are common features of
  DNA disorders. Likely due to neuronal death
  during development and in mature arrested
  neurons
• Immunodeficiency (I). The production of
  immunoglobulin and T-cell receptors requires
  the processing of double-strand breaks by the
  DNA repair machinery

Question 25

What does Base excision repair (BER) deal with ?

Answer 25

Base excision repair (BER) deals with modified bases and abasic sites produced by depurination and depyrimidination

Question 26

Explain the process of Base excision repair ?

Answer 26

1. A DNA glycosylase first cleaves the N-glycosidic bond that connects the base to the sugar (different glycosylases recognise different types of damage, for example 8-oxo-G, U). This leads to an apurinic/apyrimidinic (AP) site. Hydrolyis can also generate AP sites.
2. After the AP site is generated, incisions in the phosphodiester DNA backbone are made 5’ and/or 3’ to its side. For detail of different pathways that can lead to this, see the figure at left. A summary, which is sufficient for our purposes here, is given in the figure at right, illustrating how nucleolytic and phosphodiesterase action leads to a single nucleotide gap in one of the DNA strands.
3. In the last step DNA polymerase and ligase fill and seal this gap.

Question 27

What does Nucleotide excision repair deal with?

Answer 27

Nucleotide excision repair (NER) deals with bulky, helix-distorting DNA lesions (one example are
cyclobutane pyrimidine dimers - CPDs -, such as thiamine dimers after UV irradiation)

Question 28

Explain the process of Nucleotide excision repair?

Answer 28

1. In eukaryotes a set proteins recognises the
   damage, probably due to structural alterations
   of the double helix. This is followed by melting
   of the helix by helicases.
2. Nucleases then operate a double incision on
   either side of the damaged site, releasing a
   24-32 nucleotide long piece of single
   stranded DNA.
3. In the last step DNA polymerase and ligase fill
   and seal this gap.

Question 29

What does Mismatch repair (MMR) deal with ?

Answer 29

Mismatch repair (MMR) deals with errors in DNA replication: both mismatches and single-nucleotide indels
(hMutSα), and also short indels caused by replication slippage (hMutSβ).

Question 30

Explain the process of Mismatch repair ?

Answer 30

1. In eukaryotes a set proteins recognises the
mismatch or indel, (MSH2/6 and MSH2/3
heterodimers - see table at left).
2. The PSM2 nuclease then nicks the DNA,
and the Exo1 nuclease degrades the small
region surrounding the affected strand.
3. In the last step DNA polymerase and ligase
fill and seal this gap

Question 31

Single-strand DNA breaks (SSBs) can occur ?

Answer 31

• During DNA replication also due to oxidative attack
• If simple ligation cannot fix the break (due to complex damage to the sugar), then the damage is detected by a sensor enzyme, poly(ADP-ribose) polymerase, which modifies several protein targets and channels the repair

Question 32

If the damage involves both strands then what occurs ?

Answer 32

A double-strand break (DSB) can occur and this is then often repaired by
the non-homologous end-joining pathway (NHEJ) which only relies on the two free ends being brought together

Question 33

What is an alternative to NHEJ ?

Answer 33

A more faithful homology-dependent pathway (HDR, homology-directed repair; or sometimes HR, homologous recombination) which relies on recombination with intact DNA can be employed to repair DSBs.
This requires the presence of a homolog chromosome, or of a sister chromatid