DNA mutation and repair II

Depurination

• Sugar-base bond is spontaneously broken

• Base is lost, usually purines, and nucleotide is left empty (called apurinic site)

What would happen to apurinic site during

DNA replication?

A random base (usually adenine) is inserted, causing a mutation.

Deamination

An amino group of C or A is spontaneously removed (converted to another base)

Causes of spontaneous damage include

Depurination, deamination, oxidative damage, and transposons (aka jumping genes)

Oxidative damage

Normal process of aerobic cellular respiration creates extremely reactive atoms called free radicals

Free radicals

An atom or group of atoms that has/have an unpaired electron

Cancer and aging

Thought to be major mutagen in our cells

Transposons (aka jumping genes)

Mobile pieces of DNA abundantly found in all living things (nearly 45% of human genome)

Cut or copy themselves and then insert randomly in the host genome

• Replicative vs nonreplicative transposons

• They encode enzyme transposase

- Insertion near genes or within genes can disrupt

host gene expression

• Can also lead to larger chromosomal alterations

• DNA is being cut/pasted – can go wrong

Control transposase = control movement 

• Methylation and mRNA destruction

Some external agents (chemical and physical) can induce DNA damage:

Base analogs, alkylating agents, intercalating agents, UV light and low energy radiation, high-energy radiation (ionizing radiation), and viruses

Base analogs

Chemicals that resemble normal nucleotides and can substitute for them during DNA replication. However, they exhibit abnormal base-pairing properties

Example of a base analog

5-bromouracil resembles thymine

- DNA pol will incorporate 5BU instead of thymine during DNA rep

- 2nd round of rep – DNA pol puts a "G" across from 5BU

- 3rd round of rep – wrong "G" serves as template for wrong "C"

Alkylating agents

These chemicals add an alkyl group (CH₃ or CH₃CH₂)  to amino or ketone groups in nucleotides

Alkylated nucleotides exhibit abnormal base pairing 

Example of alkylated nucleotides

Ethyl guanine pairs with T

- Two rounds of DNA rep - Mutation 

Intercalating agents

Flat, multiple-ringed molecules that tightly wedge themselves between the bases of DNA - distorts its 3-D structure

Causes insertions or deletions in the DNA (unlike all others discussed)

UV light and low energy radiation

All electromagnetic radiation have wavelengths shorter than visible light (-380 nm) are very energetic

• Disrupt DNA and other macromolecules

UV light - λ≈260 nm and is very mutagenic

Pyrimidine dimers (usually two thymines)

UV light causes adjacent pyrimidine bases to fuse with one another (Distort DNA 3-D structure)

Pyrimidine dimers (2)

Prevents DNA pol from replicating normally

High-energy radiation (ionizing radiation) Mutates DNA in different ways:

It causes electrons to be released from various molecules in the cell producing free radicals, it directly breaks phosphodiester bonds in the DNA strands (causes double - stranded breaks), and it creates thymine dimers (Why do we treat tumors with X-rays?)

Viruses

Retroviruses have the ability to randomly insert themselves into our genome 

Example from Viruses

Retroviral gene therapy and leukemia

Other viruses produce proteins that directly inhibit

DNA replication, monitoring, or repair mechanisms

Ames test

Used to test if a new chemical has ability to mutate DNA (cause cancer)

Set-up from Ames Test

- Uses bacterial strain that can’t make its own histidine (won’t grow without it)

- Mix bacteria w/ either chemical or H 2 O

and add to Petri dish lacking histidine

- No bacteria should grow

- Mutations can occur to allow the bacteria

to make histidine - regain ability to grow

Results from Ames Test

Results H₂O control = very few colonies (spontan)

Mutagenic chemical - lots of colonies (BAD!!)

Most types of DNA damage can be fixed by the cell

Must be fixed prior to DNA replication

Exceptions that cannot be fixed by the cell

Transposons and retrovirus (can’t be removed)

Different repair mechanisms must exist to

detect and fix 

Direct DNA repair

Reserves the alteration w/o cutting out or replacing any nucleotides

Both bacteria and eukaryotic cells use

Light-dependent pathways

Eukaryotic cells

Use an enzyme called photolyase to cut abnormal covalent bonds between the two thymines

Bacteria

Use an enzyme called photoreactivation enzyme (PRE) to do the same - PRE is activated by blue light

Direct repair of alkylated bases

Methylguanine DNA methyltransferase enzymes directly cuts off extra CH₃  from guanine

Excision repair

Removal of altered base/nucleotide and replacement with

good DNA

Excision repair

1. Recognition of the lesion by 1 or more proteins and the subsequent excision of that error by a nuclease enzyme 

2. A DNA polymerase fills in the space with proper nucleotides

3. DNA ligase seals the final nick (the last phosphodiester bond) between the new and existing strands

Base excision repair

Used for correction of minor alterations to individual bases (free radical, alkylated, base analog)

Mechanism of Base Excision Repair (described in E.coli, but all cells have it)

1) DNA glycolase enzymes recognize altered based

2) Glycolase then cuts out the base only (breaking the sugar/base bond)

3) AP endonuclease enzyme recognizes the nucleotide missing the base and makes a cut in the sugar phosphate backbone at that sit

4) DNA pol I/ligase finish the job (and repair the damage)

Eukaryotic glycosylases have yet to be identifief

Examples of nucleotide excision repair

Intercalated agents, thymine dimers

NER (E.coli NER is described below)

1. DNA is damaged and a lesion forms
2. Proteins called Uvr (UvrA, B, C, D) recognize the lesion and cut it out

• A-B complex recognizes the lesion (A comes off and is replaced with C)

• A comes off and is replaced with C 

• B-C together cut the DNA on either side of the lesion (cut out extra “good genes DNA on both sides)

• D is a helicase that liberates the cut piece

3. DNA pol I fills in the gap/ ligase seals

Xeroderma pigmentosum (XP)

Contain one of several rare mutations in some part of the NER pathway

Problems of mismatch repair

If the cell has a G-T mismatch, how does it know which one is correct? (the G or the T)

- Hint #1: mismatches usually appear following DNA replication 

- Hint #2: Newly-made DNA strands stay

unmethylated for a little while

- New and old DNA strands look different for a

short time (hemi-methylated)

- If wait too long, both become methylated

- Wrong nucleotide is always on the new, unmethylated strand

Mismatch repair

DNA commonly contains methylated adenines, no effect on transcription (cytosine CH₃), and adenine methylase add CH₃ when seeing GATC

Mechanism (E. coli) of mismatch repair

1) MutS protein locates mismatches (Forms complex with MutL afterward (linker))

2) MutL binds to MutH which is bound to a nearby hemi-methylated site

3) MutH makes a cut in the unmethylated strand

4) MutU acts as a helicase to release the unmethylated strand before an exonuclease destroys it

5) DNA pol III fills in with proper sequence, ligase seals

Two repair pathways fix double-stranded breaks:

1) Homologous recombination repair (E.coli)

a) Homologous chromosome first brought in

• Usually the sister chromatid

b) RecBCD recognizes double stranded breaks

• Partially degrades 1 strand on each side

• Creates single-stranded overhangs

c) RecA binds to single-stranded end and promotes invasion of the homologous chr.

• The good strand loops up (D-loop)

d) RuvABC, DNA polymerase, and ligase help to recreate the gaps and resolve the structure

Fixing double-stranded breaks

Non-homologous end-joining

- The two broken ends are simply glued back together

together

- No requirement of sister chromatid

- End-binding proteins bind to each side of the

break (to stabilize)

- Cross-bridging proteins recruited to prevent

drifting of the two pieces

- Ends are processed, filled, and ligated

Advantage - Can happen any time in cell

cycle (no sister chr. required)

- Disadvantage - Can lead to small deletions

near the break site (result of processing)

Translesion synthesis (called SOS repair in E. coli)

- Stalling of normal DNA polymerase byu lesion triggers recruitment of "emergency" polymerases

- Have different binding pocket - more tolerant of

altered DNA structure

- Emergency pols (e.g. DNA pol II, IV, V) replicate over the lesion

    Problem: they are very error prone (DNA gets replicate, but with mistakes)

- Original lesion remains (not fixed) - transition synthesis enables rep to continue