Biochemistry for Pharmacy Students NUCLEIC ACID STRUCTURE & FUNCTION

Biochemistry for Pharmacy Students

NUCLEIC ACID STRUCTURE & FUNCTION

Pál Bauer

Dept. of Medical Biochemistry

Rm. 4515, EOK

Email: bauerpal@hotmail.com

OBJECTIVES

• To learn the structures and properties of nucleotides, the building blocks of nucleic acids

• To describe how the structure of DNA relates to functions of the genetic machinery.

• To explain how DNA is synthesized.• To describe how mutations in DNA can lead

to genetic diseases.• To explain how recombinant DNA technology

can be applied to the diagnosis and therapy of human genetic diseases (discussion sessions).

HISTORICAL PERSPECTIVE

Nucleic acid structure & metabolism is directly relevant to cancer, gout, many genetically inherited diseases, AIDS and other infectious diseases.

The beginnings of nucleic acid biochemistry & genetics occurred in the 1860s:

- Frederic Miescher – Swiss biochemist who discovered nucleic acids.

- Gregor Mendel – Austrian monk who founded the science of genetics.

Frederick Meischer studied salmon

GregorMendelstudiedgardenpeas

• Sugars and phosphates– Ribose in RNA– Deoxyribose in DNA

• Partial hydrolysis products– Nucleotides (contains

phosphates)

– Nucleosides (no phosphates)

Names of nucleosides

Base Ribonucleoside Deoxyribonucleoside

Adenine Adenosine Deoxyadenosine

Guanine Guanosine Deoxyguanosine

Cytosine Cytidine Deoxycytidine

Uracil Uridine Deoxyuridine

Thymine Thymine riboside Thymidine

Nucleosides: numbering system

• The intra-nucleotide linkage

Base composition of double stranded DNA

• [pyrimidines] = [purines]• Content: A = T, G = C• DNA base compositions are the same for

different tissues of the same organism.

Watson-Crick base pairing

Watson

Crick

Animal Cell(eukaryote)

Plant Cell(eukaryote)

Bacterial Cell(prokaryote)

A Reminder of the Differences betweenEukaryotes and Prokaryotes

Fig. 2-7

E. coli

Model of the Nuclear Envelope

Artwork by Don Guzy

• The Watson-Crick Structure

• Right-handed helices• Anti-parallel• Base-pairing• Open structure accessible

to water• Stacking forces between

planar paired bases give a rigid structure

5’ 3’

3’5’

Denaturation of DNABy pH, heat, solvents, urea, amides• Helix-coil transition• Hyperchromic effect• Melting temperature

Denaturation ofdouble-stranded

DNA

Indirect evidencea. High DNA content of chromosomesb. 260 nm is a very mutagenic wavelength;

bases maximally absorb light energy at 260 nm

c. Constancy of [DNA] / cell (germ cells) and 2x [DNA] / cell (somatic cells)

DNA IS THE GENETIC MATERIAL IN CELLS

a. Transformation of bacteria with DNA

Requires both DNA functions:

replication and expression

b. Transfection with viral nucleic acids

c. In vitro expression of DNA (from bacteriophage T4)

d. Synthesis of active DNA in vitro  

Direct Evidence

Pneumococci

DNA CONTENT

OF SOME CELLS AND VIRUSES

Haploid size of genome, Source of DNA base pairs____ Viruses

SV40 5 x 103 (5 kb)Papilloma (wart) 8 x 103

Adenoviruses 2.1 x 104

Herpesviruses 1.56 x 105

Poxviruses 2.4 x 105  Cells

Escherichia coli 4.5 x 106 (4,500 kb)Yeast 1.3 x 107

Drosophila 1.6 x 108

Human 3.2 x 109 (3.2x106 kb)Frogs 6 x 109 (6 x 106 kb)Onion Plant 18 x 109

  Fern Plant 160 x 109

Animal mitochondria 1.5 x 104 (15 kb)Plant chloroplast 1 x 105

The total length of the circular E. coli chromosome is 1.7 mm,whereas the length of an E. coli cell is 2 μm.

partially lyzedE. coli cell

  22 autosomal chromosomes (chromosomes 1-22)

plus X and Y chromosomes

3.2 x 109 total bp

Human haploid genome

• Histones and chromosome structure

a. Nucleosomes

The double-stranded DNAis wrappedaround theoutside of eachnucleosome twice.

The nucleosomesare regularly spaced along theDNA.

electron micrograph of chromatin

electron micrograph of supercoiled nucleosomesin chromatin.

A typical phone cord is coiledlike a DNA helix, and thecoiled cord can itself coil in in a supercoil. If twistedtight enough, the supercoilswill themselves form an evenhigher order of supercoiling.

Double-stranded DNA helicesalso form supercoils of

supercoils.

Two drawings depicting the differentlevels of DNA supercoiling that provideDNA compaction in a eukaryoticchromosome. The levels take the formof coils upon coils.

histones

nucleosomes

5’ 3’

DNA replication

5’ 3’

3’3’5’5’ 3’5’

5’3’

5’

5’3’

All DNA polymerases catalyze elongation ofthe primer strand in a 5’ to 3’ direction, copying the template strand in a 3’ to 5’direction. This means that the “leading strand” can be synthesized continuously, but the “lagging strand” must be synthesized discontinuously.

A problem is that DNA polymerases must have a primer strand with a 3’ OH from whichto begin DNA synthesis. So, wheredo these primers come from whena DNA polymerase synthesizes newOkazaki fragments in the laggingstrand?

5’ 3’

5’3’

5’5’ 3’3’

DNA

Replication does not usually begin at the end of a DNA molecule; itbegins in the middle of the DNA.

(DNA pol I, II and III)

12.4 Schematic model of the proofreading function of DNA

polymerase

Figure 12-21

An example of error correction by the 3’ to 5’

exonuclease (proofreading)activity of DNA polymerase I.

A mismatched base pair (a C-A mismatch) impedes the

movement of DNA polymerase Ito the next site. Sliding back-ward, the enzyme removes itsmistake with the 3’ to 5’ exo-

nuclease activity & thenresumes its 5’ to 3’

polymerase activity.

c. DNA Polymerase II

• Mutants in the E. coli gene for this DNA polymerase are not lethal. Also is a repair enzyme. • Requires duplex DNA template and primer. • Utilizes a template strand and elongates a primer

strand, similar to DNA polymerase I. d. DNA Polymerase III (pol C gene)

Mutants are ts (temperature sensitive), i.e., conditionally lethal.

The 3-D structure of E. coli DNA polymerase I.

The active site for the polymerase activity and the 3’ to 5’ exonucelase activity is deep in the crevice at the far end of the bound DNA. The template strandis dark blue.

a. Illustration of the 5’ to 3’exonuclease activity of E. coliDNA polymerase I (sometimescalled a “nick translation”activity).

An RNA or DNA strand paired with a DNA templatestrand is simultaneously degraded by the 5’ to 3’ exonuclease activity & is replaced by the polymeraseactivity of the same enzyme.

b. The origin of replication – the ori C locus Accessory proteins

dna B gene productProbably this protein is membrane-

associated and recognizes the initiation sequence on DNA.

c. RNA primersDNA polymerases require a preformed

primer; RNA polymerases do not.

1. Accessory protein binds (dna B protein) 2. "Primase," an RNA polymerase (dna G)

It does not require a primer; forms a short piece of RNA complementary to the DNA

template strand.

3. DNA binding proteins

d. Details of the events:

cellmembrane

cellmembrane

5’

5’

5’3’

5’3’

5’3’

The 5’ end of RNA primer is usually 5’ pppA... or 5’ pppG….

DNA polymerase III dissociates the primase that synthesizes the RNA primer.

The 5' → 3' nuclease of DNA polymerase l, or another enzyme called RNase H, removes the RNA primer. RNase H hydrolyzes RNA in DNA/RNA base-paired regions.

DNA polymerase I (and maybe DNA pol II?) fill the gaps left by the removal of the RNA primers.

The complete but "nicked" (broken) daughter strands that result after the gaps are filled in are then joined by DNA ligase.

Additional information about the overall process of DNA replication

• DNA ligase (animal enzyme)

• The unwinding problem: 5000 turns/min in E. coli (helicases and topoisomerases)

Topoisomerase I: relaxes supercoils

a. Enzyme recognizes right-handed superhelices

introduced during DNA replication.

b. Small DNA segment unwinds, as strand breaks, the superhelix relaxes.

c. Enzyme, still attached to broken strand, reseals the break.

d. The topoisomerase is both a nuclease and a ligase. (The nucleolytic activity can be viewed as the reverse of ligase action.)

Helicases: unwind the two strands of DNA & cause supercoiling ahead of the fork.

SUMMARY OF DNA REPLICATION IN BACTERIA

Map of the E. coli genome showing some of the genes that encodeproteins involved in DNA replication, repair and recombination.

DNA REPLICATION in EUKARYOTIC CELLS is SOMEWHATMORE COMPLICATED and INVOLVES ADDITIONAL DNA

POLYMERASES & MULTIPLE REPLICATION ORIGINS

Multiple replication origins occur in the large eukaryotic chromosomes.

Greek

Name

Gene

Name

Proposed Main Function

alpha POLA DNA Replication

beta POLB Base Excision Repair

gamma POLG Mitochondrial Replication

delta POLD1 DNA Replication

epsilon POLE DNA Replication

zeta POLZ Bypass Synthesis

eta POLH Bypass Synthesis

theta POLQ DNA Repair

iota POLI Bypass Synthesis

kappa POLK Bypass Synthesis

lambda POLL Base Excision Repair

mu POLM Non-Homologous End Joining

sigma POLS Sister Chromatid Cohesion

• The number of known eukaryotic DNA polymerases has doubled in the last several years as researchers discover additional enzymes with DNA polymerizing activity.

• Most of the newly discovered DNA polymerases are involved in repair of DNA, rather than DNA replication.

• The DNA polymerases alpha, delta and epsilon are the most clearly involved in DNA replication.

• DNA polymerases delta and epsilon have proof-reading 3’->5’ exonuclease activity; the other DNA polymerases do not.

Types and rates of mutation

Type Mechanism Frequency________ Genome chromosome 10-2 per cell division mutation missegregation

(e.g., aneuploidy)

Chromosome chromosome 6 X 10-4 per cell division mutation rearrangement

(e.g., translocation)

Gene base pair mutation 10-10 per base pair per mutation (e.g., point mutation, cell division or

or small deletion or 10-5 - 10-6 per locus per insertion generation

Mutation

Mutation rates* of selected genes

Gene New mutations per 106 gametes

Achondroplasia 6 to 40Aniridia 2.5 to 5Duchenne muscular dystrophy 43 to 105Hemophilia A 32 to 57Hemophilia B 2 to 3Neurofibromatosis -1 44 to 100Polycystic kidney disease 60 to 120Retinoblastoma 5 to 12

*mutation rates (mutations / locus / generation) can varyfrom 10-4 to 10-7 depending on gene size and whetherthere are “hot spots” for mutation (the frequency at mostloci is 10-5 to 10-6).

Many polymorphisms exist in the genome

• the number of existing polymorphisms is ~1 per 500 bp• there are ~5.8 million differences per haploid genome• polymorphisms were caused by mutations over time• polymorphisms called single nucleotide polymorphisms

(or SNPs) are being catalogued by the HumanGenome Project as an ongoing project

Types of base pair mutations

CATTCACCTGTACCAGTAAGTGGACATGGT

CATGCACCTGTACCAGTACGTGGACATGGT

CATCCACCTGTACCAGTAGGTGGACATGGT

transition (T-C to A-G) transversion (T-A to G-C)

CATCACCTGTACCAGTAGTGGACATGGT

deletionCATGTCACCTGTACCAGTACAGTGGACATGGT

insertion

base pair substitutions transition: pyrimidine to pyrimidine transversion: pyrimidine to purine

normal sequence

deletions and insertions can involve one or more base pairs

Spontaneous mutations can be caused by tautomers

Tautomeric forms of the DNA bases

Adenine

Cytosine

AMINO IMINO

Guanine

Thymine

KETO ENOL

Tautomeric forms of the DNA bases

Mutation caused by tautomer of cytosine

Cytosine

Cytosine

Guanine

Adenine

• cytosine mispairs with adenine resulting in a transition mutation

Normal tautomeric form

Rare imino tautomeric form

Mutation is perpetuated by replication

• replication of C-G should give daughter strands each with C-G

• tautomer formation C during replication will result in mispairing and insertion of an improper A in one of the daughter strands

• which could result in a C-G to T-A transition mutation in the next round of replication, or if improperly repaired

C G C G

C G C A

AC T A

Chemical mutagens

Deamination by nitrous acid

N

NH

NH

N

NH2

O

N

NH

NH

NH

NH2

O

O

Attack by oxygen free radicalsleading to oxidative damage

guanine

8-oxyguanine (8-oxyG)

• many different oxidative modifications occur• by smoking, etc.• 8-oxyG causes G to T transversions

• the MTH1 protein degrades 8-oxy-dGTP preventing misincorporation• mutation of the MTH1 gene causes increased tumor formation in mice

Ames test for mutagen detection

• named for Bruce Ames• reversion of histidine mutations by test compounds• His- Salmonella typhimurium cannot grow without histidine• if test compound is mutagenic, reversion to His+ may occur• reversion is correlated with carcinogenicity

Thymine dimer formation by UV light

Summary of DNA lesions

Missing base Acid and heat depurination (~104 purinesper day per cell in humans)

Altered base Ionizing radiation; alkylating agents

Incorrect base Spontaneous deaminationscytosine to uraciladenine to hypoxanthine

Deletion-insertion Intercalating reagents (acridines)

Dimer formation UV irradiation

Strand breaks Ionizing radiation; chemicals (bleomycin)

Interstrand cross-links Psoralen derivatives; mitomycin C

Tautomer formation Spontaneous and transient

DNA repair activity

Life

sp

an

1

10

100 human

elephant

cow

hamsterratmouseshrew

Correlation between DNA repairactivity in fibroblast cells fromvarious mammalian species andthe life span of the organism

Defects in DNA repair or replicationAll are associated with a high frequency of chromosome

and gene (base pair) mutations; most are also associated with a predisposition to cancer, particularly leukemias

• Xeroderma pigmentosum• caused by mutations in genes involved in nucleotide excision repair• associated with a >1000-fold increase of sunlight-induced skin cancer and with other types of cancer such as melanoma

• Ataxia telangiectasia• caused by gene that detects DNA damage• increased risk of X-ray• associated with increased breast cancer in carriers

• Fanconi anemia• caused by a gene involved in DNA repair• increased risk of X-ray and sensitivity to sunlight

• Bloom syndrome• caused by mutations in a a DNA helicase gene• increased risk of X-ray• sensitivity to sunlight

• Cockayne syndrome• caused by a defect in transcription-linked DNA repair• sensitivity to sunlight

• Werner’s syndrome• caused by mutations in a DNA helicase gene• premature aging

Transition vs. transversion• TransitionExchange of a purine with a purine or pyrimidine with a

pyrimindine base. More common than transversion. Often the result of tautomeric shifts.

– GC AT transition • causal agents: e.g. base analog 5’bromouracil

– AT GC transition • causal agents: e.g. base analog 2-aminopurine

• TransversionExchange of a purine with a pyrimidine base and vice versa– GC TA or GC CG transversion– AT CG or AT TA transversion

Methyl-directed mismatch repairCH3 CH3

CH3 CH3

CH3 CH3MutS

MutL

CH3 CH3

MutL

MutSMutHMutH

MutS, MutL, ATPADP+Pi

MutH, ATPADP+Pi

1. Mismatch within 1 kb ofmethylated GATC

2. MutS and MutH bind to mismatched spots along the DNA (except C-C)

3. DNA on both sides of theMitsmatch runs throughMutS:MutL complex

4. MutH binds to MutL andto GATC

5. Endonuclease of MutH cleaves unmethylated DNA at hemimethylated GATC

5’3’

5’3’

5’

5’

Mismatch repair in E. coliScenario 1Mismatch is at the 5’ end of cleavage site

1. Unmethylated DNA is unwound via DNA helicase II

2. The 3’-5’ exonuclease activity of exonuclease I or exo X degrades DNA through the mismatch

3. DNA polymerase III synthesizes the new DNA strand

4. DNA ligase closes the remaining nick.

CH3 CH3

ATPADP+Pi

MutS-MutLDNA helicase IIexonuclease I

orexonuclease X

CH3 CH3

CH3 CH3

5’3’

5’3’

5’3’

3’5’

3’5’

3’5’

DNA polymerase IIISSBs

Mismatch repair in E. coliCH3 CH3

ATPADP+Pi

MutS-MutLDNA helicase IIexonuclease VII

orRecJ nuclease

CH3 CH3

CH3 CH3

Scenario 2Mismatch is at the 3’ end of cleavage site

1. Unmethylated DNA is unwound via DNA helicase II

2. The 5’-3’ exonuclease activity of exonuclease VII or RecJ nuclease degrades DNA through the mismatch

3. DNA polymerase III synthesizes the new DNA strand.

4. DNA ligase closes the remaining nick.

5’3’

5’3’

5’3’

3’5’

3’5’

3’5’

DNA polymerase IIISSBs

Base excision repair• not restricted to a short time post replication• similar in most organisms (bacteria – mammals)• recognizes abnormal bases in the DNA• usually less expensive than mismatch repair• requires four enzymes

1. DNA glycosylases

2. AP-endonucleases

3. DNA polymerase I

4. DNA ligase

DNA glycosylases• Relatively small enzymes (20 – 30 KDa)

• Recognize abnormal bases – deaminated bases– alkylated bases

• Remove base via cleavage at the glycosidic bond between the deoxyribose and the base

• Cleavage creates apurinic and apyrimidinic (AP sites)

UOP

GOP

AOP

O

GOP

AOP

P

Before After

DNA glycosylasesEnzyme Units / mg protein (x 10 - 3)

not adapted adaptedUracil DNA glycosylase 3,800 3,500

Hypoxanthin DNA glycosylase 2.1 23- methyladenine I DNA glycosylase 3.6 3.6

3- methyladenine II DNA glycosylase (*) 0.22 4.1Formamidopyrimidine DNA glycosylase 4.2 3.9

Legend:Adapted = incubation with 1 g/ml NNG for 1 hourNot adapted = untreated control

Source: Karran et al. (1983), Nature 296, 770 - 773

AP-endonucleases• recognize AP-sites• cleave phosphodiester

bonds near the AP site and generate a 5’ –phosphate and 3’-hydroxyl

• In E. coli this enzyme also has 3’-5’ exonuclease activity

• The 3’-OH functions as a primer

P

A

P

G

P

G

P

C

P

T

P P

C

P

P

A

P

G

P

C

P

T

P

C

P

GC

P

A

P

G

P

G

P

C

P

T

P

P

A

P

G

P

C

P

C

P

GC

AP endonuclease

5’

3’

5’3’

Base excision repair (BER)

P

A

P

G

P

G

P

C

P

T

P P

C

P

G

P

A

P

G

P

G

P

C

P

T

P

U

P

C

P

G

P

A

P

G

P

G

P

C

P

T

P

G

P

A

P

G

P

G

P

C

P

T

P

C

P

C

P

G

Uracil DNA glycosylase

AP endonuclease

DNA polymerase I

DNA ligase

Defects in BER• In humans BER involves:

– DNA polymerase beta– The Xrcc1 geneproduct– Ape1 (first step in removing the damaged base)

• BER deficiencies have been implicated with: – cancer– neurodegenerative diseases– aging

Nucleotide excision repair• Recognizes large distortions in the DNA structure• Repairs UV-damaged DNA• Multisubunit enzyme • Cleaves two phosphodiester bonds upstream and

downstream of the lesion• Generally generates fragments of 12 to 13 nts• Requires four different enzymes

1. Exinuclease

2. DNA helicase

3. DNA polymerase

4. DNA ligase

Nucleotide excision repair in E. coliEnzyme Protein Function

UvrA (MW= 104,000) scans DNA, binds to UvrB

UvrB (MW = 78,000)scanner; binds DNA cleaves phosphate bond at 3' end, 5

positions downstream of lesion

UvrC (MW = 68,000) binds UvrB & DNA cleaves phosphate bond at 5' end, 8 positions upstream of lesion

DNA helicase UvrD removes DNA fragmentDNA

polymeraseDNA polymerase I

(= PolA )fills emerging gap

DNA ligase Lig seal nick

Exinuclease

Nucleotide excision repair in E. coli

exinuclease

UvrD DNA helicase

DNA pol. I DNA ligase

ATP

PP

POH

PP

UvrA UvrBUvrAMechanism

• The (UvrA)2:UvrB complex scans DNA

• UvrA dimer dissociates from pryimidine dimer. UvrB binds DNA and cuts at 3’ end.

• UvrC associates with UvrB and cuts DNA at 5’ end of the pyrimidine dimer

• UvrD DNA helicase removes the DNA fragment

• DNA polymerase I fills the gap• DNA ligase seals the remaining

nick.

Xeroderma pigmentosum• Cause:

– Defect in human nucleotide excision repair (NER)– 16 polypeptides involved in NER– NER is the only pathway to remove pyrimidine dimers

in humans

• Symptoms:– Very light sensitive– High risk of sun-light induced skin cancer– Neurological abnormalities (high rate of oxidative

metabolism in neurons)

Direct repair• Observation:

– UV-damaged bacteria that were subsequently incubated in daylight recovered better than those kept in the dark

• Photoreactivation– requires DNA photolyases– requires visible light at 300 – 500 nm– aka “light repair” – Contrast: dark repair (BER, NER, mismatch repair)

DNA photolyases• Structure

– MW = ~ 54,000 (in E. coli)– Generally contain 2 chromophores

– Chromophore No. 1: always FADH -

– Chromophore No. 2: folate (in E. coli and yeast) • N5,N10-methenyltetrahydrofolylpolyglutamate

• Function– bind to pyrimidine dimers– resolve pyrimidine dimers into original bases

Mechanism outline1. Absorption of a photon by MTHFpolyGlu

2. Transfer of excitation energy to FADH –

3. Excited FADH – transfers elecctron to the pyrimidine dimer (unstable dimer radical)

4. Shift of electrons breaks cyclobutane ring

5. Electron is transferred back to the flavin radical to regenerate FADH –

O6-methylguanine• Mutagenic behavior

– pairs with thymine rather than cytosine

• Specific Repair Mechanism – differs from base excision repair– requires O6-methylguanine methyltransferase– one time reaction (“suicide enzyme”)– expensive repair mechanism– methylated methyltransferase is a transcriptional

activator of its own gene

O6-methylguanine DNA methyltransferase

N

NN

NH2N

O-CH3

R

N

NN

NH2N

O

R

active Enz.

Cys-SH

active Enz.

Cys-S-CH3

Source: adapted from Lehninger pg. 957

O6-methylguanine nucleotide guanine nucleotide

H H

Transition vs. transversion• TransitionExchange of a purine with a purine or pyrimidine with a

pyrimindine base. More common than transversion. Often the result of tautomeric shifts.

– GC AT transition • causal agents: e.g. base analog 5’bromouracil

– AT GC transition • causal agents: e.g. base analog 2-aminopurine

• TransversionExchange of a purine with a pyrimidine base and vice versa– GC TA or GC CG transversion– AT CG or AT TA transversion

O6-methylguanine• Mutagenic behavior

– pairs with thymine rather than cytosine

• Specific Repair Mechanism – differs from base excision repair– requires O6-methylguanine methyltransferase– one time reaction (“suicide enzyme”)– expensive repair mechanism– methylated methyltransferase is a transcriptional

activator of its own gene

O6-methylguanine DNA methyltransferase

N

NN

NH2N

O-CH3

R

N

NN

NH2N

O

R

active Enz.

Cys-SH

active Enz.

Cys-S-CH3

Source: adapted from Lehninger pg. 957

O6-methylguanine nucleotide guanine nucleotide

H H

Inaccurate DNA repairE. coli recA+ strain E. coli recA- strain

Numerous survivorsHigh mutation rates

Few survivorsLow mutation rates

intensive UV exposure

Error-prone repair• Activated upon:

– severe DNA damage– disruption of DNA replication

• “SOS-response”– Inaccurate repair mechanism– Requires at least 14 proteins in E. coli

1. Din proteins (damage induced)

2. Rec poteins (recombination)

3. Umu proteins (UV-mutagenesis)

4. Uvr proteins (UV-resistance)

5. Others: SulA, HimA, Ssb, and PolB

Genes involved in the SOS response

Gene Protein function encoded:pol B DNA polymerase II (polymerization subunit)uvrA & uvrB ABC exinucleaseumuC & umuD DNA polymerase Vsul A inhibits cell division via interaction with FtsZsul BrecA recA protease, recombination and repairdinB DNA polymerase IVssb single-stranded binding proteinuvrD DNA helicase II (DNA -unwinding protein)him A subunit of integration host factorrec N required for recombinational repairdinD ?dinF ?

Damaged DNA during replication• UV-damaged DNA results in collapse of the replication fork during DNA

replication• 2 scenarios

– Unrepaired lesions Unrepaired DNA breaks

12.4 DNA damage and repair and their role in carcinogenesis

• A DNA sequence can be changed by copying errors introduced by DNA polymerase during replication and by environmental agents such as chemical mutagens or radiation

• If uncorrected, such changes may interfere with the ability of the cell to function

• DNA damage can be repaired by several mechanisms • All carcinogens cause changes in the DNA sequence and

thus DNA damage and repair are important aspects in the development of cancer

• Prokaryotic and eukaryotic DNA-repair systems are analogous

12.4 General types of DNA damage and causes

12.4 Proofreading by DNA polymerase corrects copying errors

Figure 12-20

12.4 Schematic model of the proofreading function of DNA

polymerase

Figure 12-21

12.4 Chemical carcinogens react with DNA directly or after activation, and the

carcinogenic effect of a chemical correlates with its mutagenicity

Figure 12-22

12.4 Base deamination leads to the formation of a spontaneous point

mutation

Figure 12-23

12.4 Mismatch repair of single-base mispairs

Figure 12-24

12.4 Chemically modified bases, such as thymine-thymine dimers, are corrected by

excision repair

Figure 12-25

12.4 Excision repair of DNA by the E. coli UvrABC mechanism

Figure 12-26

12.4 End-joining repair of nonhomologous DNA

Figure 12-28

12.5 Recombination between homologous DNA sites

• Recombination provides a means by which a genome can change to generate new combinations of genes

• Homologous recombination allows for the exchange of blocks of genes between homologous chromosomes and thereby is a mechanism for generating genetic diversity

• Recombination occurs randomly between two homologous sequences and the frequency of recombination between two sites is proportional to the distance between the sites

12.5 The cross-strand Holliday structure is an intermediate in recombination (part

I)

Figure 12-29

12.5 The cross-strand Holliday structure is an intermediate in recombination (part

II)

Figure 12-29