The Mutability and Repair of DNA. 1. Switch of one base for another: purine pyrimidine (transition) (transversion) 2. insertion or deletion of a nucleotide.

The Mutability and Repair of DNA

1. Switch of one base for another:

purine

pyrimidine

(transition) (transversion)

2. insertion or deletion of a nucleotide

The nature of mutations: Point mutation

Replication errors and their repair

Drastic changes in DNA

DeletionInsertionRearrangement of chromosome

By insertion of a transposon, or aberrant actions of recombinationProcess.

Some replication errors escape proofreading

Mismatch repair removes errors escape proofreading 1. It must scan the genome. 2. The system must correct the mismatch accurately.

Scan DNA

Distortion in the backbone

Embracing mismatch;Inducing a kick in DNA;Conformational change inMutS itself

(III)

Nicking is followed by Helicase (UvrD) and one of exonucleases

MutL activate MutH

DNA methylation to recognize the parental strain

Once activated, MutH selectively nicks the Unmethylated strand.

Directionality in mismatch repair

Mismatch repair system in Eukaryotics

MutS MutLE. coli

MSH(MutS homolog)

Eukaryotics MLH or PMS

Hereditary nonpolyposis colorectal cancer(mutations in human homologes of Muts and MutL)

DNA damageRadiation, chemical mutagens, and spontaneous damage

deamination

spontaneous damage due to hydrolysis and deamination

Base pair with A

depurination

Deamination converts adenine to hypoxanthine, base pair with CDeamination converts Guanine to xanthine, base with C but only two H bonds

DNA damage

spontaneous damage to generate natural base

deamination

Methylated Cs are hot spot for spontaneous mutation in vertebrate DNA

Damaged by alkylation and oxidation

Alkylation at the oxygen of carbon atom 6 of G : O6-metylguanine,often mispairs with T.

Oxidation of G generates oxoG, it can mispair with A and C. a G:C to T:A transversion is one of the most common mutation in human cancers.

Gamma radiation and X-rays

• Cause double-strand breaks in the DNA, which are difficult to repair.

• Ionizing radiation and agents like bleomycin that cause DNA to break are said to be clastogenic (p245).

DNA damage by UV

Thymine dimer

These linked bases are incapable of base-pairing and cause DNA polymerase to stop.

Mutations caused by base analogs and intercalating agents

Base analogs

Analogs mispair to cause mistakes during replication

Thymine analog

Mutations caused by intercalating agents

Intercalating agents

flat molecules

Causing addition or deletion of bases during replication

Repair of DNA Damage: DNA repair system

Excision repair systems: the damaged nucleotide is not repaired but removed from the DNA, the other undamaged strand serves as a template for reincorporation of the correct nt by DNA polymeraseRecombination repair: both strands are damaged. Sequence information is retrieved from a second undamaged copy of the chromosome.

Direct reversal of DNA damagephotoreactivation

breaking covalent bond

O6-metylguanine To its own cytosine

Capture energy from light

Base excision repairDNA glycosylases are lesion-specific and cells have multiple DNA glycosylases 1. Uracil glycosylase 2. Another specific glycosylase is responsible for removing oxoG

AP: apurinic or apyrimidinic

Base excision repair

If a damaged base is not removed by base excision before DNA replication: a fail-safe systemoxoG:A repair

Nucleotide Excision Repair

UvrA detecting distortion

UvrB melting DNA

Recognizing distortions to the shape of the DNA (thymine dimer or bulky chemical adduct)

8 nt away from 5’4-5 nt away from 3’

In E.coli: 4 proteins involved

Nucleotide Excision Repair

The principles of nucleotide excision repair in higher cells is much the same as in E. coli but us moer complicated, involving 25 or more polypeptides.The UVR proteins are needed to mend damage from UV light; Mutants of uvr genes are sensitive to UV light, and lack the capacity toremove T-T or T-C adducts.

In human, xeroderma pigmentosum patients have mutations in seven genes(XP genes). These XP proteins are corresponding to proteins involved innucleotide excision repair.

Transcription-coupled repair

Involves recruitment to the stalled RNA polymerase of nucleotide excision repair proteins

It focuses repair on genes being actively transcribed.

TFIIH unwinds the DNA template during the initiation of transcription. Subunits of TFIIH include the DNA helix-opening proteins XPA and XPD.

Translesion DNA synthesis: although are template dependent, the synthesis in a manner that is independent of base pairing

obstacles to progression of the DNA polymerase

Complex of proteins UmuC and D’

(Y-family of DNA polymerase)

(or AP site)