DNA Mutations and Repair

When do mutations occur?

1. dna replication error

2. UV/ chemical damage

3. hydrolytic reactions

When does a mispairing become solidified into the DNA

• in the FIRST ROUND OF REPLICATION= MISPAIRED

• in the second round of replication the mispaired base gets a matching base

What does UV radiation do?

FORMS A PYRIMIDINE DIMER

• carbon 6 linked to carbon 6 AND carbon 5 to carbon 5 of thymine

• pyrimidine stacked pyramid

What does nitrous acid do?

deaminates the bases

cytosine—> uracil which turns GC—> AT

N=NH2 become NH-NO

adenosine—> hypoxanthine which turns AT —> GC

mutate HXT —> replicate HXC—> replicate GC

What’s an incalating agent?

A PLANAR molecule that hops in BETWEEN 2 BASES ON THE SAME STRAND causing a new base on the bottom strand

CCC ethidium bromide TTT

GGG a nucleotide AAA

What’s a mutation caused by ethidium bromide called?

frameshift mutation

How does depurination occur?

• no base

• PDE bond intact

  • makes a sugar and phosphate inside the genetic code

Which base is easily deaminated

Cytosine (hence Uracil not inside DNA as uracil is caused by a mutation so the fire alarm can go off when uracil shows up in dna )

What is a substitution mutation?

“Point mutation” / “single base pair” causing either

1. missense mutation→ amino acid coded changed

   e.g. Val-→ Ser

2. nonsense mutation→ MAKES A STOP CODON “non more translation”

3. silent mutation→ coding region changed but amino acid is the same

Name two types of frameshift mutations

1. deletion

2. insertion

How bad is a change in one of the strands

Enzymes and proteins can REMOVED MUTATED STRAND AND USE THE UNMUTATED ONE AS A TEMPLATE= all is good in the world

How are mismatches repaired when the mismatch happens downstream of GATC where Mut H binds?

1. after replication,ONLY THE OLD STRAND IS METHYLATED

   old-CH3 vs new- :(

2. MutL and MutS complex binds to the mismatch

3. MutH binds to A on the new unmethylated GATC seq

4. MutLSH complexes making a loop

5. MutH is activated and cleaves the UNMETHYLATED STRAND on the 5’ side of GATC

6. UvrD (DNA Helicase II), unwinds unmethylated strand from the cleavage- GATC to the mismatch

exoI or exoX removes SEGMENT of unmethylated strand (3’→5’ exonuclease activity)

1. single strand binding protein stabilises the ssDNA segment while DNA Pol III adds in the bases

2. DNA ligase closes the nick

How are mismatches repaired when the mismatch happens upstream (3’ side) of GATC where Mut H binds?

1. after replication,ONLY THE OLD STRAND IS METHYLATED

   old-CH3 vs new- :(

2. MutL and MutS complex binds to the mismatch

3. MutH binds to A on the new unmethylated GATC seq

4. MutLSH complexes making a loop

5. UvrD (DNA helicase II) unwinds DNA

Exo VII or RecJ is a 5’→ 3’ exonuclease

1. SSB and DNA Pol III

2. DNA ligase

Is MutH @ eukaryotes

nope

How does base excision repair work?

• by DNA Glycosylases specific to damaged base

• Uracil-DnaGlycosylase removes U by CLEAVING N-GLYCOSIDIC BOND causing an APYRIMIDINIC SITE

• AP endonuclease recognises the baseless site and an INCISION IS MADE @ EITHER DIRECTION OF AP SITE

• DNA segment removed

• DNA POL I FILLS IN GAP

• DNA LIGASE

How does NUCLEOTIDE excision repair work?

• fixes LESIONS THAT DISTORT HELIX aka thymine dimer

• ABC exci-nuclease will HYDROLYSE THE TWO PDE BONDS THAT DISTORTS THE HELIX

• ABC exci-nuclease will REMOVE 12NTDS

• UvrA + UvrB= A2B complex

• complex binds @ lesion

• UvrA bends DNA @ lesion

• UvrC binds + UvrB

• Uvr cuts damaged DNA 8ntds upstream of dimer and 4/5 nts downstream BY USING DIFFERENT DOMAINS FOR UP/DOWNSTREAM

• UvrD releases UvrC and the dimer + ntds removed

• DNA Pol I fills gap

  • DNA Ligase

Difference between human vs E.Coli excinucleases

Human = removal of 22-29nts instead of the 5+8nts

What’s Xeroderma Pigmenntosum?

• autosomal recessive

• skin cancer risk higher

• CANT REPAIR DNA AFTER UV DAMAGE (sensitive to sun)

  • due to no excision repair ( 7 genes, jusy one needs to be mutated!)

Heriditary Non-Polypoaia Colon Cancer cause

• hereditary cancer

• autosomal DOMINANT

• can’t repair mismatches

• most commonly implicated -hMSH2 (Human Mut S homolog 2) -hMLH1 (Human Mut L homolog 1)