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Lecture 4 Mutations & DNA Repair

DNA damaging agents

Mutagen
Spontaneous

Mechanism
DNA replication; repair errors, spontaneous modification of nucleotides Pyrimidine dimers induce error prone repair (SOS) Base analog Base analog Alkylating agent, generates O6methyl guanine Alkylating agent, generates N4hydroxycytosine DNA intercalating agent Ionization of molecules into free radicals and reactive ions

Type of mutation
All types of mutations

UV irradiation 2-aminopurine (2-AP) Bromouracil Ethylmethane sulfonate (EMS) & N-methyl-N'-nitroN-nitrosoguanidine (MNNG) Hydroxylamine (NH2OH) Acridine dyes (acridine orange, proflavin) Ionizing radiation (X-rays, rays, cosmic rays)

Mainly G-C to A-T transitions A-T to G-C and G-C to A-T transitions G-C to A-T and A-T to G-C transitions G-C to A-T transitions

G-C to A-T transitions when used in vitro Frameshift probably due to slippage during replication Point mutations; disrupts integrity of chromosomes

Ames Test
To assess the mutagenicity of compounds
Filter paper containing ethyl methanesulfonate (EMS)

Growth of his+ revertants

Classes of Mutations

DNA damage produced by chemical mutagens fall into 2 major classes


1.

Point mutations
a) Transitions, in which a purine (or pyrimidine) is replaced by another b) Transversions, in which a purine is replaced by a pyrimidine or vice versa

2.

Insertion/deletion mutations

Missense mutation due to a base substitution


Met Thr Asp Glu - ATG ACC GAC GAG - TAC TGG CTG CTC - Met Thr Glu Glu - ATG ACC GAA GAG - TAC TGG CTT CTC - -

Silent mutation due to a base substitution


Met Thr Glu Glu - ATG ACC GAG GAG - TAC TGG CTC CTC - Met Thr Glu Glu - ATG ACC GAA GAG - TAC TGG CTT CTC - -

Insertion mutation
Met Thr Asp Glu - ATG ACC GAC GAG - TAC TGG CTG CTC - Met Thr Asp Arg Arg Glu - ATG ACC GAC CGA CGA GAG - TAC TGG CTG GCT GCT CTC - -

Deletion mutation
Met Thr Asp Glu - ATG ACC GAC GAG - TAC TGG CTG CTC - Met Asp Glu - ATG GAC GAG - TAC CTG CTC - 7

Point mutations are generated by altered bases

Nitrous acid (HNO2) oxidatively deaminate primary amines Hence, A=T GC and GC A=T transitions Nitrite (the conjugate of HNO2) is used as a preservative in prepared meats

Alkylating agents such as dimethyl sulfate, nitrogen mustard, ethylnitrosourea, and MNNG generates transitions
DNA exposed to MNNG yields O6-methylguanine residues which can base pair with either C or T.

Insertion/Deletion mutations are generated by intercalating agents

The distance between 2 consecutive base pairs is doubled by the intercalation of such molecules Replication of such DNA results in deletion or insertion of one or more nucleotides in the newly synthesized DNA Frameshift mutation
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Frameshift insertion mutation


Met Thr Asp Glu - - Met Lys ATG ACC GAC GAG - - ATG AAA TAC TGG CTG CTC - - TAC TTT -

Nonsense mutation (chain termination)


Met Thr Asp Glu - - Met Lys ATG ACC GAC GAG - - ATG AAA TAC TGG CTG CTC - - TAC TTT -

Met Thr Arg Arg - ATG ACC CGA CGA G - TAC TGG GCT TCT C - -

Met Thr Glu Stop - ATG ACC GAC TAG - - ATG AAA TAC TGG CTG ATC - - TAC TTT

Note: Frameshift mutation can also arise due to deletion

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Spontaneous alterations likely to require DNA repair

1. Oxidative damage 2. Hydrolytic attack 3. Uncontrolled methylation S-adenosyl methionine


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Depurination and deamination due to hydrolytic attack


OR ADENINE

If left uncorrected, such changes could lead to deletion or substitution of base pairs during DNA replication
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Deamination of bases in DNA yields an unnatural base which can be directly recognized and removed by a specific DNA glycosylase E.g. deamination of C produces U, which can be repaired by uracil DNA glycosylase

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DNA mispairing also occurs when methylated C is converted to T by deamination

G:C base pair

G:T base pair

About 3% of C nucleotides in vertebrates are methylated to help in controlling gene expression Accidental deamination of 5-methyl-cytosines yields thymines, thus resulting in mismatched base pairing
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Deaminated and depurinated nucleotides may result in nucleotide substitutions or deletions


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DNA damage induced by mutagens


Thymine or pyrimidine dimers

Formation of a dimer between 2 pyrimidine bases when cells are exposed to UV irradiation Occurs between two adjacent thymine or cytosine bases Repaired by nucleotide excision repair system
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Frameshift deletion due to slipped strand mispairing

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DNA Repair Systems

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DNA Damage

Mutations Replication errors Replication Persistent DNA damage Genomic instability

Cancer

DNA Repair

Aging

DNA repair .

A major defense against environmental damage to cells Minimizes cell killing, mutations, replication errors, persistence of DNA damage and genomic instability Abnormalities in these processes have been implicated in cancer and aging
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Types of damage in DNA

Spontaneous damage
Depurination Deamination

Damage induced by mutagens


Thymine dimers Substitutions Deletions/insertions Frameshift mutations Double-strand breaks
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Several types of DNA Repair systems in bacteria


Repair of DNA synthesis errors
A. Proofreading by DNA polymerase B. Mismatch repair by mutHSL

Repair of DNA modifications


C. Direct reversal of damage- Photoreactivation repair D. Excision repair by DNA glycosylase and AP endonuclease
Base excision repair Nucleotide excision repair

Repair of replication fork barriers


E. Translesion synthesis

Repair of breaks in DNA


F. Repair of DSB by homologous and non-homologous end joining
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Errors arise during DNA during replication

Complementary base pairing alone is not sufficient to determine fidelity Rare tautomeric forms of the 4 bases (A, T, G, C) occur transiently (1:104 or 1:105). Rare tautomeric form of C can also pair with A

Proofreading by DNA polymerase decreases errors introduced during DNA synthesis by 1000-fold Strand-directed mismatch repair reduces the errors by a further 100-fold

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(A) Proof-reading by DNA Polymerase

DNA Pol has a higher affinity for correct nucleotide because of base pairing requirement 35 proofreading exonuclease activity of DNA Pol removes mismatched nucleotide at 3OH end of primer strand

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(B) Strand-directed Mismatch repair

MutS binding to DNA Singlestrand Gap

During DNA replication, mismatch repair corrects errors that remain after proof-reading In eukaryotes, newly synthesized DNA are recognized by presence of many nicks Defects in the human mismatch repair system result in a high incidence of cancer e.g. hereditary nonpolyposis colorectal cancer

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Mismatch Repair in E. coli


In E. coli, mismatch repair involves the MutSLH system Depends on the methylation of selected A residues in GATC to distinguish newly synthesized vs template DNA An endonuclease makes a nick on 5 side of the unmethylated GATC UvrD (helicase) + exonuclease removes defective strand The unmethylated DNA strand is corrected by DNA Pol III

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(C) Direct Reversal of Damage Photoreactivation repair

Removes DNA modification (thymine dimers) Light-dependent reaction Occurs in bacteria but not in eukaryotes Involves a photoreactivation enzyme (PRE) cleaves bonds between thymine dimers
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(D)

Excision Repair

Occurs in both prokaryotes & eukaryotes 2 types


Base excision repair (BER) involving DNA glycosylases specific to each type of altered base e.g. deaminated bases Nucleotide excision repair (NER) for bulky lesions e.g pyrimidine dimers, mutagen cross-linked DNA

Involves 3 steps
Recognition & removal of distortion or error by a nuclease Gap filling by DNA polymerase Sealing of nick by DNA ligase
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Base Excision Repair (BER)


Removes damaged base Many types of DNA glycosylases, each of which cleaves the glycosidic bond leaving a deoxyribose residue with no attached base - apurinic or apyrimidinic sites (AP sites) Most common is uracil-DNA glycosylase which removes uracil (arising from spontaneous deamination of cytosine)
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Nucleotide Excision Repair (NER)

UvrABC endonuclease

Corrects pyrimidine dimers and other DNA lesions in which the bases are displaced from their normal position or have bulky substituents. In E. coli, NER is an ATP-dependent process involving UvrA, UvrB, UvrC and UvrD proteins In humans, NER requires >16 proteins. Individuals with xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are unable to repair UV-induced DNA lesions XP individuals have 2000-fold incidence of skin cancer
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UvrD helicase

Repair of Thymine Dimers

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(E) Translesion DNA synthesis

When a lesion is encountered during replication, Pol III is replaced by error-prone a translesion DNA polymerase, Pol IV (DinB) or Pol V (UmuD2C) Translesion DNA polymerase extends DNA synthesis beyond thymine dimer independent of base pairing and has no proofreading exonuclease activity

Pol III

Translesion DNA polymerase replaces Pol III

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(contd)

Pol III then replaces translesion DNA Pol to complete DNA replication Translesion DNA synthesis is error-prone. In E. coli, translesion polymerases are invoked as a last resort as part of the SOS response Individuals with a variant form of XP exhibit are defective in translesion DNA synthesis. They have normal NER proteins but show increased rates of skin cancer

Pol III

Pol V

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The E. coli SOS response activates error-prone repair

In response to DNA damage, RecA triggers the SOS system and induces 43 genes involved in recombinational repair Proteolytic activity of
DNA Pol IV and V are error prone translesion polymerases UvrABCD are involved in NER
Cleavage of LexA repressor induces the expression of many target genes

LexA recA

Other target genes

activated RecA cleaves LexA repressor

DNA damaging agents activates RecA

Other target uvrB, genes lexA recA

uvrA, uvrC, uvrD

LexA, RecA and other target genes involved in general homologous recombination and recombinational repair are induced

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Recombination repair of double strand breaks


2 different ways to repair DSBs:

emergency repair
Figure 5-51 Molecular Biology of the Cell ( Garland Science 2008)

Non-homologous end-joining (NHEJ)

DSB repair by nonhomologous end joining (NHEJ) is common in mammalian somatic cells Ku is a key protein in NHEJ

Figure 5-52a Molecular Biology of the Cell ( Garland Science 2008)

Homologous end-joining

Repair of double-strand break in DNA by homologous end-joining

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Double strand break is generated when the replication fork encounters a singlestrand nick in the template DNA DSBs are also induced by ionizing radiation, replication errors, oxidizing agents and certain cellular metabolites

DSB is generated

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Homologous strand exchange initiated by double strand break

Occurs predominantly in mitotic cells

Occurs predominantly in meiotic cells

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Inherited syndromes with defects in DNA repair


NAME MSH2, 3, 6, MLH1, PMS2 Xeroderma pigmentosum (XP) Cockayne syndrome PHENOTYPE Colon cancer Skin cancer, cellular UV sensitivity, neurological abnormalities Hypersensitivity to UV, stunted growth, neurological dysfunction, premature aging Breast and ovarian cancer Premature aging, cancer at several sites, genome instability Cancer at several sites, stunted growth, genome instability Congenital abnormalities, leukemia, ENZYME OR PROCESS AFFECTED Mismatch repair Nucleotide excision repair Nucleotide excision repair Repair by homologous recombination Accessory 3exonuclease and DNA helicase Accessory DNA helicase for replication DNA interstrand crosslink repair 42

BRCA-2 Werner syndrome

Bloom syndrome Fanconi anemia gps A-G

Reading

Alberts et al. Molecular Biology of the Cell 5th Ed. (2008), Chapter 5, p295-304 Voet & Voet, Fundamentals of Biochemistry 2nd Ed (2006), Wiley Chapter 24

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