lecture 09 10 [compatibility mode]

8/3/2019 Lecture 09 10 [Compatibility Mode]

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MUTATIONS AND

DNA REPAIR


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Mutations alter the DNA sequences

Mutations are heritable alterations in the genetic material of any organism

Single strand of normal -globin gene Single strand of mutant -globin geneGTGCACCTGACTCCTG AGGAG GTGCACCTGACTCCTG TGGAG

Single nucleotide change (mutation)

-Single nucleotide change produces a -globin that

differ from normal -globin only by a changefrom Glu to Val at thesixth amino acid position. -The mutation causes thedisease sickle-cell anemia


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The major types of mutation

Base substitution : one base is replaced by another base Insertion : one or more bases are inserted into the DNA sequence Deletion : one or more basesare deleted from the DNA sequence Inversion : a segment of DNA is

inverted, but remains at the same overall location

Duplication : a segment of DNA is duplicated; the second copy usuallyremains at the same location as the original

Translocation : a segment of DNA is transfer from its original location to

another position either on the same DNA molecule or on the different DNAmolecule


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The nature of mutations

Transitions : pyrimidine-to-pyrimidine and purine-to-purine substitutions, such as T to C and A to G

Transversions : pyrimidine-to-purine and purine- to-pyrimidine substitutions, such as T toA or G and A to T or C

- Base substitutions (transitions and transversions), Insertions and Deletionsare simple mutations. Other kinds of mutations might be caused by transposon or recombination process

- Mutations that alter a single nucleotide are called point mutations


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Slippage during DNA replication creates

small insertions or deletions

I n human, slippage of trinucleotide (CGG and CAG) repeats causes disease(fragile X syndrome and Huntingtons disease)


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Some Inherited Syndromes with Defects in DNA Repair


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Sources of mutation

I naccuracy in DNA Chemical damage toreplication the genetic material

Consequences1) permanent changes to the DNA (mutation)2) prevent DNAs use as a template for replication and

transcription

* The cell must scan the genome to detect error in synthesis anddamage to the DNA;

** The cell must mend the lesions do so in that way that, if possible,restores the original DNA sequence.


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REPLICATION ERROR ANDTHEIR REPAIR


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Proofreading of DNA synthesis

- DNA synthesis : approximately 1 mistake in every 10 10 base pair added.

Proofreading allows these mistakes to be corrected.- DNA polymerase has an error-correcting activity ( proofreading exonuclease ).- I n palm domain, there are DNA replication site (P) and Exonuclease site(E) . - When a mismatched base pair is detected, the primer : template juntion slidesaway from P site and into E site. After the incorrect base pair is removed, the

primer : template junction slides back in to P site and DNA synthesis can continue.


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Some replication errors escape proofreading

I f the misincorporated nucleotide is not detected and replaced, the sequencechange will become permanent (mutation) in the genome after second roundreplication.


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DNA mismatch repair system removes errorsthat escape proofreading


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Mismatch repair pathway for the repair of replication errors

MutS scans the DNA anddetects mismatch

- MutL activates MutH , an enzyme that cause a nick onone strand near the site of mismatch-A specific helicase (UrvD) unwinds the DNA, and theexonuclease progressively digest the mismatch region- The gap is then filled by DNA polymerase and sealedwith DNA ligase.


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Crystal structure of the MutS-DNA complex


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How does the cell know which of the twomispaired bases was the wrongone?

- In E. coli , the parental strand of DNA isidentified by methylation.

-The enzyme Dam methylase convertsAdenine in the sequence GATC toMethyladenine

- Immediately after DNA replication, theold strands will be methylated but thenew strands of DNA will not ( Hemi-methylated )

- The newly synthesized strand ismarked (non-methylated) and is checkedfor errors before methylation occurs


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Directionality in mismatch repair:

exonuclease removal of mismatched DNA

Different exonucleases are used to remove single stranded DNA between the nick created by MutH and the mismatch, depending on whether MutH cuts the DNAon the 5 or 3 side of the misincorporated nucleotide.


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Repair mismatch system in eukaryotes

-In eukaryotic cells also repair mismatch and

do so using homologs to MutS (called MSH proteins for MutS homologs) and MutL(called MLH and PMS ).

- Eukaryotes have multiple MutS-like proteins with different specificity . For example, one is specific for simple mismatch,whereas another recognize small insertions or deletions resulting from slippage -Eukaryotes lack MutH and do not have hemi-methylation. MSH interacts with the slidingclamp and recruits mismatch repair proteins.


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DNA DAMAGE


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A summary of spontaneous alterations likely to require DNA repair

The sites that are known to be modified by oxidative damage

Hydrolytic attack

Uncontrolled methylation


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Depurination and deamination are the most frequentchemical reaction known to create serious DNA damage

in cells

- Depurination : purine bases (A and G) will be lost from DNA - Deamination :loss of an amino group from cytosine to produce the base uracil


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How chemical modifications of nucleotides produce mutations


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Deamination


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DNA is damaged by alkylation, oxidation

-In alkylation : methyl or ethyl groups aretransferred to reactive sites on the bases andto phosphate in the DNA backbone -Oxidation of guanine generates oxoG(common mutations found in human cancers)


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Thymine dimer: UV induces the formation of a cyclobutanering between adjacent thymines

These linked bases are incapable of base-pairing and cause the DNA polymeraseto stop during replication


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Mutations are also caused by base analogs


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Mutations are also caused by intercalating agents

- Intercalating agents are flat molecules containing several polycyclic rings that bind to the equally flat purine or pyrimidine bases of DNA, just as the bases bindor stack with each other in the double helix

- Intercalating agents cause the deletion or addition of a base pair or, even few base pairs


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REPAIR OF DNA DAMAGE


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Photoreactivation cleaves thymine dimer

I n photoreactivation, the enzyme DNA photolyase captures energy from light anduses it to break the covalent bonds linking adjacent pyrimidine. The damaged

bases are mended directly


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Direct reversal of methylated base

The enzyme methyltransferase removes the methyl group from the guanineresidue by transfering it to one of its own Cys residues.


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Base excision repair

- An enzyme called glycosylase recognizesand removes the damaged base byhydrolyzing the glycosidic bond.

- Glycosylase release the base from theDNA backbone to leave an AP site (apurinicor apyrimidic site)

- AP endonuclease and exonuclease cut theDNA backbone at the 5 and 3 position of the AP site

- The resulting gap is filled by DNA polimerase I

- DNA glycosylase are lesion- specific andcell have multiple DNA glycosylase withdifferent specificities. A total8 differentDNA glycosylase have been identified inhuman cells.


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oxoG:A can be repaired via the base excision pathway


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Structure of a DNA-glycosylase complex


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Nucleotide excision repair

UvrC forms a complexwith UvrB and createsnicks to the 5 and 3 sidesof the lesion

UvrA and UvrB scan theDNA to identify a

distortion

UvrB melt locally aroundthe distortion


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Transcription-coupled DNA repair

- RNA polymerase transcribes DNAnormally upstream of the lesion

- Upon encoutering the lesion in DNA,RNA polymerase stalls and transcriptionstops

- RNA polymerase recuits the nucleotideexcision proteins to the site of the lesion andrepairs

- Central to transcription-coupled repair ineukaryotes is the general transcriptionfactor TF II H


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Double-strand break (DSB) repair pathway


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DSB repair pathway: Non homologous end joining

Three-dimensional structure of a Kuheterodimer bound to the end of aduplex DNA fragment


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Translesion DNA synthesis enables replication to proceedacross DNA damage

- If cells cannot repair lesions, there is afail-safe mechanism that allow thereplication machinery to bypass thesesites of damage. This mechanism isknown as translesion synthesis. -Translesion synthesis is catalyzed by aspecialized class of DNA polymerase(UmuC or Y-family DNA polymerase)


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DNA repair systems

lecture 09 10 [compatibility mode]

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