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� Mutation in germ line would destroy the species

� Mutation in soma would destroy the

individual.

Maintenance of the correctness of the DNA sequence is definitely crucial for living organisms. Keeping the error rate as low as 10-10 is so expensive.

ErrorsErrorsErrorsErrors� Inaccuracy Inaccuracy Inaccuracy Inaccuracy in DNA in DNA in DNA in DNA replicationreplicationreplicationreplication

� Chemical Chemical Chemical Chemical damage to the genetic material (damage to the genetic material (damage to the genetic material (damage to the genetic material (environment)environment)environment)environment)� Lesions Lesions Lesions Lesions (arose from spontaneous damage) (arose from spontaneous damage) (arose from spontaneous damage) (arose from spontaneous damage) � Damage Damage Damage Damage (caused by chemical agents and radiation(caused by chemical agents and radiation(caused by chemical agents and radiation(caused by chemical agents and radiation

� First, Detect the First, Detect the First, Detect the First, Detect the errorserrorserrorserrors

� Second, Mend/repair the errors or lesions in a way Second, Mend/repair the errors or lesions in a way Second, Mend/repair the errors or lesions in a way Second, Mend/repair the errors or lesions in a way to restore the original DNA sequence.to restore the original DNA sequence.to restore the original DNA sequence.to restore the original DNA sequence.

To repair an error or damageTo repair an error or damageTo repair an error or damageTo repair an error or damage

Replication errorsReplication errorsReplication errorsReplication errors

The nature of mutations

Point mutations:

1. Transitions (pyrimidine to pyrimidine, purine to purine)

2. Transversions (pyrimidine to purine, purine to pyrimidine)

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Insertions

Deletions

Gross rearrangement of chromosome.

These mutations might be caused by insertion by These mutations might be caused by insertion by These mutations might be caused by insertion by These mutations might be caused by insertion by These mutations might be caused by insertion by These mutations might be caused by insertion by These mutations might be caused by insertion by These mutations might be caused by insertion by transposontransposontransposontransposontransposontransposontransposontransposon or by aberrant action of cellular or by aberrant action of cellular or by aberrant action of cellular or by aberrant action of cellular or by aberrant action of cellular or by aberrant action of cellular or by aberrant action of cellular or by aberrant action of cellular recombination processes.recombination processes.recombination processes.recombination processes.recombination processes.recombination processes.recombination processes.recombination processes.

Rate of spontaneous mutation at any given site on chromosomal ranges from 10-6 to 10-11 per round of DNA replication, with some sites being “hotspot” .

Mutation-prone sequence in human genome are repeats of simple di-, tri- or tetranucleotide

sequences, known as DNA microsatellites.

These sequences:

(1) are important in human genetics and disease,

(2) hard to be copied accurately and highly

polymorphic in the population.

� Expansions of repeated DNA sequences represent a newly discovered type of human mutation.

� Such mutations and their phenotypic consequences occur when many extra copies are made of three basepair DNA sequences normally found in low copy number in or near genes.

� Fragile X mental retardation (involves multiple replications of the nucleotide repeat CGG )

� Myotonic Dystrophy (expansion involving excessive copying of CTG repeat)

� Huntington Disease (autosomal dominant, was found to result from expansion of a triplet repeat (CAG) )

Some replication errors escape proofreading

The 3’-5’ exonuclease activity of replisome only improves the fidelity of DNA replication by a

factor of 100-fold.

The misincorporated nucleotide needs to be

detected and replaced, otherwise it will cause mutation.

Generation of MutationGeneration of MutationGeneration of MutationGeneration of Mutation

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Mismatch repair removes errors that

escape proofreading

Increase the accuracy of DNA synthesis for 2-3 orders of magnitudes.

Two challenges:

(1) rapidly find the mismatches/mispairs,

(2) Accurately correct the mismatch

DNA damageDNA damageDNA damageDNA damageMutations arise not only from errors in replication, but also from damage to the DNA.

Environment factors – radiation and so-called mutagens (chemical agents)

Spontaneous damage from action of water

Most common hydrolytic damage is deamination of the base cytosine, generating the unnatural base uracil in DNA.

Uracil pairs with adenine, and so introduces that base instead of guanine as directed by cytosine.

Deamination of adenine to hypoxanthine (pairs with cytosine instead of thymine).

Deamination of guanine to xanthine (pairs with cytosine but only two hydrogen bonds)

Deamination C→→→→U

DNA also undergoes depurination by spontaneous hydrolysis of N-glycosyl linkage, and this produces an abasic site

(deoxyribose lacking a base) in the DNA.

Hydrolysis creates apurinic deoxyribose

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DNA is damaged by alkylation, oxidation

and radiation

In alkylation, methyl and ethyl groups are transferred to reactive sites on the bases and to phosphates in the DNA backbone.

DNA is subjected to attack by Reactive Oxygen Species (ROS) and UV light. UV radiation causes the photochemical fusion of two

pyrimidines that occupy the adjacent positions on the same polynucleotide chain. In the case

of two thymines, the fusion is called thymine dimers, which

contains a cyclobutane ring.

Gamma radiation and X-rays (ionizing radiation) cause double-strand breaks and are particularly hazardous (hard to be repaired).

Ionizing radiation can directly attack the deoxyribose in the

DNA backbone, or generating ROS.

Mutations are also caused by base analogs and intercalating

agents.�Base analogs: similar enough to the normal bases to be

processed by cells and incorporated into DNA during replication. �But they base pair differently, leading to mistake during

replication.�The most mutagenic base anolog is 5-bromouracil.�Intercalating agents: flat molecules that interact with the normal bases in DNA through hydrogen bonds and base stacking.

Can cause deletions and additions in genes, thus shifting the coding sequence out of range.

Base analogs

Intercalating agents

� Some damages, such as thymine dimer, nick or breaks in the DNA backbone, create impairments to replication or transcription

� Some damages creates altered bases that has no effect on replication but cause mispairing, which in turn can be converted to mutation.

Recombination repair/

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Mismatch repair

To repair mismatched bases, the system has to know which base is the correct one.

In E. coli, this is achieved by a special methylase called the "Dam methylase", which can methylate all adenines that occur within (5')GATC sequences.

Immediately after DNA replication, the template strand has been methylated, but the newly synthesized strand is not methylated yet. Thus, the template strand and the new strand can be distinguished.

In E.coli, MutS/MutL and MutH are essential for DNA

mismatch repair.

Eukaryotic cells also repair mismatches and do so using homologs to MutS(MSH) and MutL (MLH). The underlying mechanisms are not the same and not well understood.

� MutS, also known as the "mismatch recognition" enzyme, is essential for the DNA mismatch repair biological pathway.

� It recognizes base-base mismatches and small nucleotide insertion/deletion mispairs generated during DNA synthesis or damage caused by various agents.

� MutS scans the DNA, recognizing the mismatch from the distortion they cause in the DNA backbone

� MutS embraces the mismatch-containing DNA, inducing a pronounced kink in the DNA and a conformational change in MutS itself

�� The The repairing process begins with the protein repairing process begins with the protein MutSMutS which which binds binds toto mismatched base pairs.mismatched base pairs.

�� ThenThen, , MutLMutL is recruited to the complex andis recruited to the complex and activates activates MutHMutHwhich which binds to GATC sequences.binds to GATC sequences.

�� Activation Activation of of MutHMutH cleaves the cleaves the unmethylatedunmethylated strand at the strand at the GATC GATC site.site.

�� SubsequentlySubsequently, the segment from the cleavage site to the , the segment from the cleavage site to the

mismatch mismatch is removed by is removed by exonucleaseexonuclease (with assistance from (with assistance from helicasehelicase and and SSB proteins).SSB proteins).

�� If If the cleavage occurs on the 3' side of the mismatch, this the cleavage occurs on the 3' side of the mismatch, this step step is carried out by is carried out by exonucleaseexonuclease I (which degrades a single I (which degrades a single strand strand only in the 3' to 5' direction).only in the 3' to 5' direction).

�� If the cleavage occurs on the 5' side of the mismatch, If the cleavage occurs on the 5' side of the mismatch,

exonucleaseexonuclease VII or VII or RecJRecJ is used to degrade the single is used to degrade the single stranded DNA.stranded DNA. The gap is filled by DNA polymerase III and The gap is filled by DNA polymerase III and DNA DNA ligaseligase..

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Different exonucleases are used to remove ssDNAbetween the nick created by MutH and the

mismatch. If GATC is located on 5’ of mutation If GATC is located on 3’ of mutation

Base excision

• DNA's bases may be mutated by deamination or alkylation.• The position of the modified (damaged) base is called the "abasic site" or "AP site".• In E.coli, the DNA glycosylasecan recognize the AP site and

remove its base.• Then, the AP endonucleaseremoves the AP site and

neighboring nucleotides.• The gap is filled by DNA polymerase I and DNA ligase.

Nucleotide excision

In E. coli, proteins UvrA, UvrB, and UvrC are involved in

removing the damaged nucleotides (e.g., the dimer

induced by UV light).The gap is then filled by DNA polymerase I and DNA ligase.

In yeast, the proteins similar to Uvr's are named RADxx ("RAD" stands for "radiation"), such as RAD3, RAD10. etc.

� Photoreactivation

� Methyltransferase action

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Photoreactivation

PhotoreactivationPhotoreactivation directly reverses the formation of directly reverses the formation of pyrimidinepyrimidinedimersdimers that result from UV irradiation. that result from UV irradiation.

The enzyme DNA The enzyme DNA photolyasephotolyase captures energy from light and uses captures energy from light and uses it to break the covalent bonds linking the adjacent it to break the covalent bonds linking the adjacent pyrimidinespyrimidines, , and mending the and mending the DNA directly.DNA directly.

Methyltransferase

Direct removal of the methyl group from the methylated O6-methylguanine. The methyl group is transferred to the protein

itself, inactivating the protein. Costly!!

TranslesionTranslesionTranslesionTranslesionpolymerase and polymerase and polymerase and polymerase and process process process process

Translesion DNA synthesis enables replication to proceed across DNA damage

� Error-prone repair***� Occurs when the above repairs are not efficient

enough so that a replicating polymerase encounters a lesion

� Translesion synthesis is also called a fail-safe or last resort mechanism.

1. Translesion synthesis is catalyzed by a specialized class of DNA polymerases that synthesize DNA directly across the damage site.

2. Translesion polymerase is produced by cell in

response to the DNA damage3. Translesion polymerases are expressed as part of

the SOS response pathway.

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Translesion DNA synthesis

Upon encountering a lesion in the template during replication, DNA polymerase III with its sliding clamp dissociates from the DNA, and is

replaced with translesionpolymerase, which extends DNA synthesis across the

thymine dimer.

Translesion polymerase is then replaced by DNA polymerase III.