DNA damage and repair Flashcards
what is the difference between DNA damage and DNA mutations?
- Mutations: change in the sequence of bases without structure alterations - they are NOT corrected by DNA repair mechanisms
- Damage: structure DNA modification with bond alterations - ARE recognised by DNA repair mechanisms
3 classifications of mutations
- point
- deletion/insertion
- frameshift
point mutation definition:
a change in a single base either via:
transition: purine to purine
transversion: swap between purine-pyrimidine
3 effects of a point mutation
- silent - no alteration of amino acids due to genetic code being degenerate
- missense - a single amino acid is changed
- nonsense - altered codon corresponds to a STOP codon so the protein is shorter in length
Deletion mutation def
a mutation in which a part of the DNA sequence is left out during replication -> any number of nucleotides can be erased this way
insertion mutation def
type of mutation involving addition of extra genetic material of varying sizes
Frameshift mutation
mutations in which deletion or insertion of a nucleotide sequence that is not a multiple of 3 causes the alteration of the entire translational frame following the mutation –> very severe effects if it occurs in coding region
what factors can cause a mutation?
- spontaneous mistakes during DNA replication (escaped by DNAP proofreading activity) –> this causes MISMATCH
- mutagenic effect (environmental) which react with DNA and modify the structure –> this causes changes in nucleotides and dimerisation
what is mismatch?
-when the base pairs are not complementary due to an error in replication (point mutation) - exists due to the presence of tautomers
How do tautomers affect mismatch?
TAUTOMERS: isomers that purines and pyrimidines can exist in which have different proton states
-create rare base pairing:
rare A binds with common C, and rare T binds with common G
HENCE: the error made during replication is not because DNAP doesn’t respect base complementarity, but because there is a temporary shift of a common base to a rare base and hence mismatching occurs
Categories of mutagens (3)
- physical
- chemical
- biological
Action of physical mutagens
Ionising radiation: cause DNA double stranded breakage
UV radiation: induces pyrimidine dimerisation
Heat: breaks glycosidic bonds (but less severe than other two)
Chemical mutagens action
alkylating agents: add alkyl groups on nucleobases
base analogues -> Bromouracil!! causes transition of A to G
intercalating agents: insert themselves between nucleobases cause frameshift mutations –> eg. ethidium bromide
biological mutagen types
viruses
bacteria
transposons (chromosomal segments that can undergo transposition)
Different existing types of damage that can be done on the DNA - bases and backbone
BASES:
-deamination
-hydrolysis of N-glycosidic bonds
-oxidation
-methylation/alkylation
BACKBONE:
-hydrolysis of phosphodiester bonds
3 systems to reduce chance of mutation from replication
-high accuracy of DNAP
-exonuclease proofreading activity
-DNA repair mechanisms being triggered
classifications of DNA repair mechanisms (4)
- direct reversal: immediate and direct repair of DNA damage
- excision repair: removes damaged portion of DNA (single base or a portion) and replaces it with a new one
- DNA mismatch repair: corrects DNA replication mistakes (mismatches)
- DNA break repair: repair of breaks on single and/or double strands
Direct reversal process
- use of DNA ligase to fill in single stranded nicks (missed spots) from replication
- fixes mistakes in methylation/alkylation
EUK: facilitated by MGMTs (methyl guanine methyl transferases) –> transfer of CH3 from guanine to MGMT cysteine residues which inactivates the enzyme (suicide enzymes)
PROK: facilitated by Ada –> CH3 are transferred from bases to Ada which changes it into a TF –> it then induces transcription of genes that help cell fight alkylation damage
- Reverses pyrimidine dimerisation: visible light activates photolyase enzymes, which then uses FADH- to repair UV-induced lesions. (NOT IN HUMANS ONLY IN BACTERIA)
2 types of excision repair
BER - base er
NER - nucleotide er
BER trigger and mechanism
USED FOR: non-bulky lesions for damage caused by oxidation/ionising radiation/ alkylation damage
MECHANISM:
1. recognition of damage by the DNA glycosylase enzyme
2. elimination of base via cleavage of N-glycosylated bond
3. generates AP site (site with no base present)
4. APE1 (endonuclease) cleaves phosphodiester backbone
5. gap is filled using DNA ligase
2 types of BER mechanisms
- short patch: removal of one nucleotide and occurs using:
DNAP beta and DNA ligase 3 - long patch: removal of 2-8 nucleotide portions involving:
DNAP delta/epsilon, Ligase 1
NER trigger and mechanism
USED FOR: removal of BULKY damage usually UV induced
(found in all organisms)
2 categories:
-GG NER: global genomic: controls entire genome
-TC NER: transcription coupled: acts on regions transcribed by RNAP2
MECHANISM:
1. DNA legion recognised in GG NER occurs using XPC (and TF2H recruitment) while in the TC NER recognition occurs using CSA/CSB
2. endonucleases cut out sequence
3. RNAP fills the gap and DNA ligase 3 seals it
Human pathologies that can arise from mutations in NER proteins
Xeroderma
pigmentosum and
Cockayne’s syndrome (due to use of XPC, CSA and CSB)
Mismatch repair trigger and mechanism (MMR)
USED FOR: repair of wrong insertions/deletions and mis-incorporated nucleotides
MECHANISM: (in pro)
-acts DURING replication to distinguish new strand from template strand to check accuracy of replication:
1.MutS protein binds to mismatched base pairs and induces a distortion of DNA
2.MutL binds to MutS /DNA complex and recruits MutH
- MutH (an endonuclease recognizes the daughter DNA strand which is not methylated, splits it at nearest GATC sites and excises the DNA strand
- DNA polymerase, ligase and methylase complete the daughter strand
!MutH not present in euk so nicks drive the process
How does the MMR process distinguish between the parent and daughter strands during replication?
-in PROK: newly synthesised strand is recognised because it is unmethylated whereas parent strand is
- in EUK: newly synthesized strand is identified due to presence of occasional nicks
what are the two types of break repairs
- single strand BR
- double strand BR
Pathology stemming from mutation of MMR proteins
Lynch syndrome -> colon cancer
ssBR trigger and process
USED FOR: repairs to single DNA strand errors resulting from oxidising agents, ionizing rad, nuclear enzymes
MECHANISM:
1. PARP1 protein detects damage
2. adds poly ADP-ribose to target proteins which recruits other proteins (XRCC1)
3. missing DNA synthesised by DNAP and ligase closes nicks
what are the 2 types of DSBR?
- NHEJ: non homologous end joining
- HR: homologous recombination
DSBR trigger
USED FOR: double strand breaks (eg. stemming from ionising rad)
DSBR NHEJ mechanism
NHEJ:
1. Recognition of double strand breaking by the antigen Ku
2) Recruitment and activation of DNA phosphokinase
3) End joining using ligase 4 to seal the gap
! Ku is a dimer made of Ku70 and Ku80 proteins that are each wrapped in a ring around the DNA ends to prevent exposure to endonucleases
DSBR HR mechanism
-occurs during meiosis to allow crossing over and requires a homologous chromosome as well as DNA double helix
MECHANISM:
1. 5’ end is degraded so 3’ end is longer
2. replication bubble formed by the invasion of 3’ to other sister chromatid
3. separation of strands on double helix
4. pairing of 3’ ends with strands of homologous chromosome
5. terminals are elongated by DNAP
6. resolution of junction
pathologies arising from mutations to the DSBR proteins
BRCA1 and BRCA 2 can cause ovarian cancer
what are the cell’s responses to DNA damage
Cell cycle is arrested to allow repair of DNA, and if this is not possible apoptosis follows
