protecting the genome

Question 1

what are free radicals?

Answer 1

byproduct of normal metabolism in human cells that sometime react with DNA and cause chemical changes

Question 2

what are the most common free radicals? how is t formed?

Answer 2

reactive oxygen species generated most often by the incomplete reduction of oxygen during mitochondrial oxidative phosphorylation

Question 3

at what wavelength does DNA absorb UV light most efficiently? shorter wavelengths=? are shorter wavelength highly damageable to DNA? which UV light is most damaging to our DNA?

Answer 3

260nm
higher energy
yes
UVB

Question 4

what are other types of radiation?

Answer 4

X-rays and radioactive elements

Question 5

what are ionising radiation (IR)? how do they affect DNA?

Answer 5

penetrating radiation that causes DNA damage

damages DNA either directly or by generating ROS from water

Question 6

what are exogenous chemicals that cause DNA damage?

Answer 6

environmental pollutants
natural toxins
dietary chemicals
anti-cancer drugs

Question 7

how does mechanical damage arise?

Answer 7

mechanical damage are endogenous physical damage that arise from errors in chromosome replication or segregation causing chromosome to be torn apart by the mitotic spindle apparatus

Question 8

what are mechanical DNA damage?

Answer 8

failure of controlled replication origin firing may cause re-replication in the centromeric region resulting in one sister chromatid with 2 centromeres. depending on how the spindles attach, the outcome may be chromosome mis-segregation or physical breakage of the chromosomal DNA

Question 9

what are types of biological damage?

Answer 9

viruses –> insert their genomes into the genome of their host cells
DNA replication errors that result from nucleotide disincorporation or replication slippage
chromosome segregation failure (=2 sister chromatid will move to a single pole)

Question 10

much of endogenous genome damage are unpreventable and must e repaired by cellular mechanisms. what are these mechanisms?

Answer 10

3 mechanisms:

• neutralising reactive oxygen species (ROS)
• avoiding DNA replication errors
• avoiding chromosome segregation errors

Question 11

how do cells avoid DNA replication errors?

Answer 11

DNA polymerase of replisome, during elongation sense nucleotide mis-incorporation and use their 3’- to 5’- exonuclease activity to remove the offending nucleotide before resuming synthesis

Question 12

what is the spindle assembly checkpoint?

Answer 12

mechanism that prevents aberrant chromosome segregation at anaphase and any resulting aneuploid or chromosome breaks
it uses specialised proteins in the kinetochore to sense the spindle tension

Question 13

what is depurination?

Answer 13

removal of a purine base (A or G) from DNA by hydrolysis, leaving an basic site (=no base)
it blocks replication and transcription and needs repairing by base excision repair

Question 14

what is deamination?

Answer 14

removal of an amino group
if happens to cytosine, cytosine becomes uracil and uracil in DNA is thought to block DNA rep and must be repaired by base excision repair
if happens to 5-methyl-cytosine, it becomes thymine which unless removed becomes a fixed mutation in the DNA sequence

Question 15

what is mismatch repair?

Answer 15

if DNA polymerase missed a few mistakes a 2nd set of proteins correct the errors
it involves recognition of the affected region, followed by removal and re-synthesis of one DNA strand, using the parental strand as template
it must distinguish between the newly synthesised strand with the error from the parental strand

Question 16

how does mismatch repair work?

Answer 16

MutSa and MutLa (repair enzymes) recognise base mismatches
they then diffuse away from the mismatch to reach nicks in the newly synthesised strand bound by either PCNA or RFC
exonuclease (EXO1) is then recruited to degrade the newly synthesis strand towards and through the mismatching region
PCNA then recruits a DNA polymerase to re-synthesise the gaped region, leaving nicks to be sealed by DNA ligase

Question 17

what is base excision repair?

Answer 17

repair mechanism that repairs the DNA with damages or missing bases or with a single strand breaks

Question 18

how does base excision repair work?

Answer 18

DNA glycosylase identifies and removes the damaged base by cleaving the glycosyl bond between the base and DNA backbone
endonuclease then cuts the sugar-phosphate backbone
the baseless sugar-phosphate is removed
and DNA polymerase fills the gap using the undamaged strand as a template and DNA ligase seals it

Question 19

what is nucleotide excision repair?

Answer 19

removes DNA damage that distorts the DNA double helix

Question 20

on what depends the way the damage is recognised?

Answer 20

depends on wether it is in a region transcribed by RNApIII (transcription coupled NER) or elsewhere in the genome (global NER)

Question 21

what are the steps of NER?

Answer 21

XPC recognises damaged DNA to initiate the global repair pathway
DNA is unwound around the damage region by helicases
endonuclease make an incision several nucleotides upstream and downstream of the damaged region
25-30 nucleotides are excised around the damage region. DNA is re-synthesised and sealed by DNA polymerase and DNA ligase

Question 22

what is the Cockayne’s syndrome (CS)?

Answer 22

disease associated with defective NER and is characterised by short stature and neurological dysfunction and mild skin sensitivity
it is an autosomal recessive disorder caused by mutations in the genes for CSA or CSB proteins which have roles in transcription coupled repair which is required for post-translational modification of RNApolII

Question 23

what are the 2 most common pathways for repairing double strand breaks?

Answer 23

homologous recombination and non-homologous end-joining

Question 24

what does homologous recombination do?

Answer 24

uses undamaged section of similar DNA as template
enzymes interlace the damage and undamaged strands to get them to exchange sequences of nucleotides and finally fit in the missing gaps to end up with 2 complete double stranded segments

Question 25

what is homologous recombination?

Answer 25

important part of meiosis by generating genetic diversity by forming crossover products between chromosome homologue
homologous recombination also occurs in mitosis where it acts as a double strand break repair mechanism by using the intact sister chromatid as a template

Question 26

is homologous recombination more or less accurate than non homologous en joining? why?

Answer 26

more accurate because it uses a template

Question 27

when is homologous recombination inactive? why?

Answer 27

during G1 because that is when the sister chromatids are not available

Question 28

when can NHEJ occur?

Answer 28

at anytime during the cell cycle

Question 29

is NHEJ error-prone?

Answer 29

yes

Question 30

why is NHEJ important in adaptive immunity?

Answer 30

it mediates the rearrangement of the immunoglobulin and T cell rector genes
that is why inherited mutations in the genes encoding certain NHEJ proteins can cause various inherited immunodeficiency syndromes

Question 31

what are sensor proteins?

Answer 31

detect DNA damage and with the help of mediators, recruit and activate transducer kinases ATM and ATR

Question 32

what are transducers kinases?

Answer 32

phosphorylate effector kinase Chk1 and Chk2 that phosphorylate effector kinase Chk1 and Chk2 that phosphorylate further effector proteins that control transcription factors, apoptosis and cell cycle arrest

Question 33

why are cancerous cells more susceptible to agents used in radiotherapy and chemotherapy?

Answer 33

because they divide more frequently than normal cells