Lecture 4 - Mutation & DNA Repair

8/3/2019 Lecture 4 - Mutation & DNA Repair

1/42

1

Lecture 4

Mutations & DNA Repair


2/42

2

DNA damaging agents


3/42

3

Mutagen Mechanism Type of mutation

Spontaneous DNA replication; repair errors,spontaneous modification of

nucleotides

All types of mutations

UV irradiation Pyrimidine dimers induce errorprone repair (SOS)

Mainly G-C to A-T transitions

2-aminopurine (2-AP) Base analog A-T to G-C and G-C to A-Ttransitions

Bromouracil Base analog G-C to A-T and A-T to G-Ctransitions

Ethylmethane sulfonate(EMS) & N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Alkylating agent, generates O6-methyl guanine

G-C to A-T transitions

Hydroxylamine (NH2OH) Alkylating agent, generates N4-

hydroxycytosine

G-C to A-T transitions when used

in vitroAcridine dyes (acridineorange, proflavin)

DNA intercalating agent Frameshift probably due toslippage during replication

Ionizing radiation (X-rays, -rays, cosmic rays)

Ionization of molecules into freeradicals and reactive ions

Point mutations; disrupts integrityof chromosomes


4/42

4

Ames Test

To assess themutagenicity ofcompoundsFilter paper containing

ethyl methanesulfonate

(EMS)

Growth ofhis+

revertants


5/42

5

Classes of Mutations


6/42


7/42

7

Met Thr Glu Glu - -

ATG ACC GAG GAG - -

TAC TGG CTC CTC - -

Met Thr Glu Glu - -

ATG ACC GAAGAG - -TAC TGG CTT CTC - -

Silent mutation dueto a base substitution

Met Thr Asp Glu - -

ATG ACC GAC GAG - -

TAC TGG CTG CTC - -

Met Thr Glu Glu - -

ATG ACC GAAGAG - -TAC TGG CTT CTC - -

Missense mutation due

to a base substitution

Met Thr Asp Glu - -

ATG ACC GAC GAG - -

TAC TGG CTG CTC - -

Met Asp Glu - -

ATG GAC GAG - -

TAC CTG CTC - -

Deletion mutation

Met Thr Asp Glu - -

ATG ACC GAC GAG - -

TAC TGG CTG CTC - -

Met Thr Asp Arg Arg Glu - -

ATG ACC GAC CGA CGAGAG - -

TAC TGG CTG GCT GCT CTC - -

Insertion mutation


8/42

8

Point mutations are generated byaltered bases

Nitrous acid (HNO2)oxidatively deaminateprimary amines

Hence, A=T GCand GC A=Ttransitions

Nitrite (the conjugate

of HNO2) is used as apreservative inprepared meats


9/42

9

Alkylating agents such as dimethyl sulfate, nitrogenmustard, ethylnitrosourea, and MNNG generatestransitions

DNA exposed to MNNG yields O6-methylguanine residues whichcan base pair with either C or T.


10/42

10

Insertion/Deletion mutations aregenerated by intercalating agents

The distance between 2consecutive base pairsis doubled by theintercalation of suchmolecules

Replication of such DNA

results in deletion orinsertion of one or morenucleotides in the newlysynthesized DNA Frameshift mutation


11/42

11

Met Thr Asp Glu - - Met Lys -

ATG ACC GAC GAG - - ATG AAA -

TAC TGG CTG CTC - - TAC TTT -

Met Thr Asp Glu - - Met Lys -

ATG ACC GAC GAG - - ATG AAA -

TAC TGG CTG CTC - - TAC TTT -

Met Thr Glu Stop - -ATG ACC GAC T AG - - ATG AAA

TAC TGG CTGATC - - TAC TTT

Nonsense mutation (chaintermination)

Met Thr Arg Arg - -ATG ACC CGA CGA G - -

TAC TGG GCT TCT C - -

Frameshift insertionmutation

Note: Frameshift mutation can also arise due to deletion


12/42

12

Spontaneous alterations likely torequire DNA repair

1. Oxidative damage

2. Hydrolytic attack

3. Uncontrolled methylation S-adenosyl methionine


13/42

13

Depurination and deamination due tohydrolytic attack

OR ADENINE

If left uncorrected, such changes could lead to deletion or substitutionof base pairs during DNA replication


14/42

14

Deamination of bases inDNA yields an unnatural

base which can bedirectly recognized andremoved by a specificDNA glycosylase

E.g. deamination of Cproduces U, which can berepaired by uracil DNA

glycosylase


15/42

15

DNA mispairing also occurs when methylatedC is converted to T by deamination

About 3% of C nucleotides in vertebrates are methylatedto help in controlling gene expression

Accidental deamination of 5-methyl-cytosines yieldsthymines, thus resulting in mismatched base pairing

G:C base pair G:T base pair


16/42

16

Deaminated and depurinated nucleotides may result innucleotide substitutions or deletions


17/42

17

DNA damage induced by mutagens

Thymine or pyrimidinedimers

Formation of a dimer

between 2 pyrimidinebases when cells areexposed to UV irradiation

Occurs between two

adjacent thymine orcytosine bases

Repaired by nucleotideexcision repair system


18/42

Frameshift deletion due toslipped strand mispairing

18
http://f/TEACHING/LSM2102/KL%20Chua/2009-10/Movies/Slipped%20strand%20mispairing%20&%20frameshift%20deletion.swf


19/42

19

DNA Repair Systems


20/42

20

Mutations

Replication errors

Persistent DNA

damage

Genomic

instability

Replication

DNA Damage

DNA Repair

Cancer

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 cancerand aging


21/42

21

Types of damage in DNA

Spontaneous damage

Depurination

Deamination

Damage induced by mutagens

Thymine dimers

Substitutions

Deletions/insertions

Frameshift mutations

Double-strand breaks


22/42

22

Several types of DNA Repair systemsin 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 barriersE. Translesion synthesis

Repair of breaks in DNA

F. Repair of DSB by homologous and non-homologous end joining


23/42

23

Errors arise during DNA duringreplication

Complementary base pairing alone is not sufficient todetermine 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 errorsintroduced during DNA synthesis by 1000-fold

Strand-directed mismatch repair reduces the errorsby a further 100-fold


24/42

24

DNA Pol has a higher affinity for correct nucleotidebecause of base pairing requirement

35 proofreading exonuclease activity of DNA Polremoves mismatched nucleotide at 3OH end of primer

strand

(A) Proof-reading by DNA Polymerase


25/42

25


26/42

26

(B) Strand-directed Mismatch repair

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 highincidence of cancer e.g. hereditary nonpolyposis colorectal

cancer

MutS binding toDNA

Single-

strand Gap


27/42

27

Mismatch Repair in E. coli

In E. coli, mismatch repair involvesthe MutSLH system

Depends on the methylation ofselected A residues in GATC todistinguish newly synthesized vs

template DNA

An endonuclease makes a nick on5 side of the unmethylated GATC

UvrD (helicase) + exonuclease

removes defective strand The unmethylated DNA strand is

corrected by DNA Pol III


28/42

28

(C) Direct Reversal of Damage -Photoreactivation repair

Removes DNA modification(thymine dimers)

Light-dependent reaction

Occurs in bacteria but not ineukaryotes

Involves a photoreactivationenzyme (PRE)

cleaves bonds betweenthymine dimers


29/42

29

(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.gpyrimidine 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


30/42

30

Base Excision Repair (BER)

Removes damaged base

Many types of DNAglycosylases, each of whichcleaves the glycosidic bondleaving a deoxyribose

residue with no attachedbase - apurinic orapyrimidinic sites (AP sites)

Most common is uracil-DNA

glycosylase which removesuracil (arising fromspontaneous deamination ofcytosine)


31/42

31

Nucleotide Excision Repair (NER)

Corrects pyrimidine dimers and

other DNA lesions in which thebases are displaced from theirnormal position or have bulkysubstituents.

In E. coli, NER is an ATP-dependent

process involving UvrA, UvrB,UvrC and UvrD proteins

In humans, NER requires >16proteins.

Individuals with xeroderma

pigmentosum (XP) and Cockaynesyndrome (CS) are unable to repairUV-induced DNA lesions

XP individuals have 2000-fold incidence of skin cancer

UvrABC endonuclease

UvrD helicase


32/42

Repair of Thymine Dimers

32
http://f/TEACHING/LSM2102/KL%20Chua/2009-10/Movies/Thymine%20Dimers.swf


33/42

(E) Translesion DNA synthesis

When a lesion isencountered duringreplication, Pol III isreplaced by error-prone atranslesion DNA

polymerase, Pol IV (DinB)or Pol V (UmuD2C)

Translesion DNApolymerase extends DNA

synthesis beyond thyminedimer independent of basepairing and has noproofreading exonuclease

activity 33

Pol III

Translesion DNApolymerasereplaces Pol III


34/42

(contd) Pol III then replaces translesion

DNA Pol to complete DNAreplication

Translesion DNA synthesis iserror-prone. In E. coli,

translesion polymerases areinvoked as a last resort as partof the SOS response

Individuals with a variant formof XP exhibit are defective intranslesion DNA synthesis.They have normal NERproteins but show increasedrates of skin cancer

34

Pol IIIPol V

Th E li SOS i


35/42

35

The E. coliSOS response activates error-pronerepair

In response to DNAdamage, RecA triggers theSOS system and induces43 genes involved inrecombinational repair DNA damaging agents

activates RecA

LexA

recA

Othertarget genes

Proteolytic activity of

activated RecA cleavesLexA repressor

Cleavage of LexArepressor inducesthe expression ofmany target genes

LexA, RecA and other targetgenes involved in generalhomologous recombinationand recombinational repairare induced

uvrA,uvrB,

uvrC,uvrD

Other targetgenes

lexA

recA

DNA Pol IV andV are error pronetranslesion

polymerases UvrABCD are

involved in NER

Recombination repair of double strand


36/42

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

Recombination repair of double strandbreaks

emergency repair

2 different ways to repair DSBs:

N h l d j i i (NHEJ)


37/42

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

Non-homologous end-joining (NHEJ)

DSB repair by non-

homologous end joining(NHEJ) is common inmammalian somatic cells

Ku is a key protein in

NHEJ


38/42

39

Homologous end-joining

Repair of double-strandbreak in DNA byhomologous end-joining


39/42

40

Double strand break is

generated when thereplication forkencounters a single-strand nick in thetemplate DNA

DSBs are also induced byionizing radiation,replication errors,oxidizing agents andcertain cellularmetabolites

DSB is generated


40/42

41

Occurspredominantlyin mitotic cells

Occurspredominantlyin meiotic cells

Homologous strand exchange

initiated by double strand break

Inherited syndromes with defects in


41/42

42

Inherited syndromes with defects inDNA repair

NAME PHENOTYPE

ENZYME OR PROCESS

AFFECTED

MSH2, 3, 6, MLH1,PMS2

Colon cancer Mismatch repair

Xerodermapigmentosum (XP)

Skin cancer, cellular UV sensitivity,neurological abnormalities

Nucleotide excisionrepair

Cockayne syndrome Hypersensitivity to UV, stuntedgrowth, neurological dysfunction,premature aging

Nucleotide excisionrepair

BRCA-2 Breast and ovarian cancer Repair by homologousrecombination

Werner syndrome Premature aging, cancer at several

sites, genome instability

Accessory 3-

exonuclease and DNAhelicase

Bloom syndrome Cancer at several sites, stuntedgrowth, genome instability

Accessory DNA helicasefor replication

Fanconi anemia gps A-G Congenital abnormalities, leukemia, DNA interstrand cross-link repair


42/42

Reading

Alberts et al. Molecular Biology of the Cell 5thEd. (2008), Chapter 5, p295-304

Voet & Voet, Fundamentals of Biochemistry 2nd

Ed (2006), Wiley Chapter 24

Documents

Lecture 4 - Mutation & DNA Repair