DNA Mutations,

Damage & Repair

Lecture 4

Cellular DNA Repair Responses

I. Damage Excision
Base excision Repair (BER)
Nucleotide excision Repair (NER)
Mismatch Repair

Nucleotide Excision Repair

UvrA and UvrB protein recognizes damaged DNA
distortion of double helix.
UvrA is released and UvrB remains, UvrC is recruited to the
site of the lesion.
UvrC+UvrB cleaves several bases away on the 3 and 5
sides respectively, flanking the damaged site, excising a ~1215 oligonucleotide.

Nucleotide Excision Repair

UvrD (helicase) removes the damage-containing

oligonucleotide from the dsDNA molecule and the

resulting gap is filled by DNA polymerase I and sealed

by ligase.
https://www.youtube.com/watch?v=rYreAcn04-M

Nucleotide Excision Repair

UvrABC Exinuclease System
UvrA

UvrB

Recognises DNA damage

Effect of caffeine on incision step of

nucleotide excision repair

Nucleotide Excision Repair &

Coupling with transcription
DNA damage blocks transcription: transcription-repair
coupling is advantageous by allowing the cell to
preferentially repair damage to actively expressed genes.
RNA polymerase stalled at a damaged site in the DNA
strand being transcribed is recognised by a protein called
transcription-repair coupling factor - displaces RNA
polymerase and recruits the UvrABC excinuclease system
to the site of damage.
UvrABC system removes the error and the transcription
continues.

Nucleotide Excision Repair &

Coupling with transcription

Methyl-directed Mismatch Repair

DNA repair in the absence of external damage

Mismatch repair
Bases on both strands have correct structure but are
mismatched.

The proof reading activity of DNA polymerase does not

always correct mismatched base pairs.

Back-up repair for replication errors

How does cell recognise the correct base?

Methyl-directed Mismatch Repair

Mismatch recognition
-

strand discrimination

GATC sequence on parental DNA

strand is hemi-methylated by DAM
methylases with Adenine methylated
at C6

long patch repair

Dam Methylation

Dam Methylase

Proof that DNA methylation is needed

for mismatch recognition
dam mutants

DAM methylase -ve

Result = mutations increase
Reason = no strand discrimination

DAM methylase overproduction

Results = mutations increase
Reason = No time for strand discrimination

Methyl-directed Mismatch Repair

Methyl-directed Mismatch Repair

E. coli MutHLS System consist of 3 main proteins

MutS scans the DNA for mismatches and binds to the
mismatch
MutH and MutL bind to the MutS protein to form a
complex

The complex spools DNA stopping at the nearest

methylated GATC parental sequence - MutH nicks the nonmethylated daughter strand at GATC.
Exonucleases remove nucleotides from the non-methylated
cut daughter strand including the mismatch.
DNA Polymerase III fills in the gaps using the methylated
parental strand followed by ligation to seal the end.

Methyl-directed Mismatch Repair