MBB 331 Lecture 8

What are the 3 major classes of mutations

Point mutations

Indels

Large-scale mutations

What is a point mutation

A change of one nucleotide to another nucleotide

How does a point mutation become permanent

First a mismatch occurs. If the mismatch is not repaired before the next round of replication, it becomes a permanent

What is an indel

An insertion or deletion of nucleotides

What are the two types of indels

Frameshift indels and In-frame indels

What is a frameshift mutation

An insertion or deletion that is not a multiple of 3 nucleotides, causing the reading frame to shift

Why are frameshift mutations usually severe

can change every codon downstream of the mutation and can completely disrupt the protein

What is an in-frame mutation

An insertion or deletion of nucleotides in multiples of 3, so the reading frame stays intact

What can an in-frame mutation do to a protein

Add or remove amino acids while maintaining the reading frame

What causes triplet expansion diseases

DNA slippage during replication causes repeated insertion of triplet sequences

What is a transition mutation

type of point mutation where one nitrogenous base is replaced by another base of the same chemical class

What is a transversion mutation

point mutation where a DNA base is replaced by a base from a different chemical class

Which bases are purines

A and G

Which bases are pyrimidine

C and T

is C → T a transition or transversion

Transition (pyrimidine → pyrimidine)

Which type of mutation is more common

Transitions because purines have the same overall shape

What is a synonymous (silent) mutation

mutation that changes the codon but does NOT change the amino acid

What is a non-synonymous mutation

mutation that changes the amino acid sequence

What is a missense mutation

One amino acid is replaced by another amino acid

What is a nonsense mutation

A mutation that creates a premature stop codon

What is deamination

Removal of an amino group from a base

Removal of an amino group from a base

Cytosine → Uracil (C → U)

What causes deamination

spontaneous hydrolysis (most common) or chemicals such as HNO₂

Why are CpG sites mutation hotspots

5-methylcytosine deaminates to thymine, producing C→T mutations

What is depurination

Loss of a purine base (A or G) from DNA

What is produced during depurination

An AP (abasic) site

What mutations can depurination cause

Point mutations or deletions

What causes oxidative damage

Reactive oxygen species (ROS)

Name the major ROS

Hydrogen peroxide, hydroxyl radicals, superoxide radicals

What mutation can 8-oxoG cause

GC → TA transversion

What is alkylation

Addition of an alkyl group to DNA

Common alkylation targets

N3 of adenine and O6 of guanine

What happens when guanine is alkylated at O6

It pairs with T instead of C, causing GC → AT mutation

What does UV radiation cause

Pyrimidine dimers (especially thymine dimers)

Why are thymine dimers harmful

They distort DNA and prevent normal base pairing

What do gamma rays and X-rays cause

Single-strand and double-strand DNA breaks

What are the different mechanisms for repair

Direct repair

Base excision repair

Nucleotide excision repair

Mismatch repair

What is direct repair

Damage is repaired on-site without removing the nucleotide

Which enzyme repairs O6-methylguanine

O6-methylguanine methyltransferase

What happens to the methyltransferase enzyme after repair

It becomes permanently inactivated

Which enzyme repairs thymine dimers by direct repair

DNA photolyase

What happens if protein is missing

No methyltransferase → alkylation mutations accumulate

No photolyase → thymine dimers accumulate

What damage does BER repair

Small damaged bases such as deaminated or oxidized bases

First BER enzyme

DNA glycosylase

What does DNA glycosylase do

Removes damaged base and creates AP site

What does AP endonuclease do

Cuts DNA at the AP site

What does DNA Pol I do

Replaces removed DNA

What does ligase do

Seals the nick

BER If Protein Missing

No glycosylase → damage never detected

No AP endonuclease → AP site accumulates

No Pol I → gap remains

No ligase → nick remains

What damage does NER repair

Bulky DNA lesions such as thymine dimers

Which proteins find damage in E. coli NER

UvrA and UvrB

Which protein cuts both sides of damage

UvrC

Which protein removes damaged fragment

UvrD helicase

Which proteins finish repair

DNA Pol I and DNA ligase

What happens if protein is missing

No UvrA/UvrB → lesion not found

No UvrC → lesion not excised

No UvrD → damaged piece not removed

No ligase → nick remains

What does MMR repair

Replication mismatches and small indels

How does E. coli know which strand is wrong

New strand is not yet methylated

Which protein recognizes mismatch

MutS

Which protein works with MutS

MutL

Which protein cuts the unmethylated strand

MutH

Which proteins remove damaged DNA

Helicase II and exonuclease

Which proteins fill and seal the gap MMR

DNA Pol III and ligase

What happens if protein is missing

No MutS → mismatch not recognized

No MutL → repair complex fails

No MutH → wrong strand not cut

No helicase/exonuclease → mismatch not removed

No Pol III/ligase → repair unfinished

O6-alkylguanine damages what's the repair

Direct Repair

Thymine Dimer damages what's the repair

Direct Repair or NER

Deamination, Oxidation, and AP Site damages what's the repair

BER

Bulky Distortion damages what's the repair

NER

Replication Mismatch damages what's the repair

MMR