DNA Replication, Mutation, and Repair

a). DNA replicationi). Cell cycle/ semi-conservative replicationii). Initiation of DNA replicationiii). Discontinuous DNA synthesisiv). Components of the replication apparatus

b). Mutationi). Types and rates of mutationii). Spontaneous mutations in DNA replicationiii). Lesions caused by mutagens

c). DNA repairi). Types of lesions that require repairii). Mechanisms of repair

Proofreading by DNA polymeraseMismatch repairExcision repair

iii). Defects in DNA repair or replication

The mammalian cell cycle

G1

S

G2M

G0

DNA synthesis and histone synthesis

Growth and preparation forcell division

Rapid growth and preparation forDNA synthesis

Quiescent cells

phase

phase

phase

phase

Mitosis

DNA replication is semi-conservative

Parental DNA strands

Daughter DNA strands

Each of the parental strands serves as a template for a daughter strand

origins of DNA replication (every ~150 kb)

replication bubble

daughter chromosomes

fusion of bubbles

bidirectional replication

Origins of DNA replication on mammalian chromosomes

5’3’

3’5’

5’3’

3’5’

3’5’

5’3’

Initiation of DNA synthesis at the E. coli origin (ori)

5’3’

3’5’

origin DNA sequence

binding of dnaA proteins

A A A

dnaA proteins coalesce

DNA melting inducedby the dnaA proteinsA

AA

AA

A

AA

AA

A

A B C

dnaB and dnaC proteins bind to the single-stranded DNA

dnaB further unwinds the helix

A

A

A

AA

A B C

dnaB further unwinds the helix and displaces dnaA proteins

GdnaG (primase) binds...

A

A

A

AA

AB C

G...and synthesizes an RNA primer

RNA primer

B C

G

5’ 3’template strand

RNA primer(~5 nucleotides)

Primasome dna B (helicase) dna C dna G (primase)

OH3’ 5’

3’

5’ 3’

RNA primer

newly synthesized DNA

5’

5’

DNA polymerase

Discontinuous synthesis of DNA

3’5’

5’ 3’

3’ 5’

Because DNA is always synthesized in a 5’ to 3’ direction,synthesis of one of the strands...

5’3’ ...has to be discontinuous.

This is the lagging strand.

5’3’

3’5’

5’3’

3’5’

5’ 3’

3’ 5’

5’3’

3’5’

5’3’

leading strand (synthesized continuously)

lagging strand (synthesized discontinuously)

Each replication fork has a leading and a lagging strand

• The leading and lagging strand arrows show the direction of DNA chain elongation in a 5’ to 3’ direction• The small DNA pieces on the lagging strand are called

Okazaki fragments (100-1000 bases in length)

replication fork replication fork

RNA primer

5’3’

3’5’

3’5’

direction of leading strand synthesis

direction of lagging strand synthesis

replication fork

5’3’

3’5’

3’5’

Strand separation at the replication fork causes positivesupercoiling of the downstream double helix

• DNA gyrase is a topoisomerase II, which breaks and reseals the DNA to introduce negative supercoils ahead of the fork• Fluoroquinolone antibiotics target DNA gyrases in many gram-negative bacteria: ciprofloxacin and levofloxacin (Levaquin)

5’3’ 5’

3’

Movement of the replication fork

Movement of the replication fork

RNA primerOkazaki fragment

RNA primer

5’

3’

RNA primer5’

DNA polymerase III initiates at the primer andelongates DNA up to the next RNA primer

5’

5’3’

5’

newly synthesized DNA (100-1000 bases) (Okazaki fragment)

5’3’

DNA polymerase I inititates at the end of the Okazaki fragment and further elongates the DNA chain while simultaneously removing the RNA primer with its 5’ to 3’ exonuclease activity

pol III

pol I

newly synthesized DNA (Okazaki fragment)5’

3’

5’3’

DNA ligase seals the gap by catalyzing the formationof a 3’, 5’-phosphodiester bond in an ATP-dependent reaction

5’3’

3’5’

Proteins at the replication fork in E. coli

Rep protein (helicase)

Single-strandbinding protein (SSB)

BC

G Primasome

pol I

pol III

pol III

DNA ligase

DNA gyrase - this is a topoisomerase II, whichbreaks and reseals double-stranded DNA to introducenegative supercoils ahead of the fork

Components of the replication apparatus

dnaA binds to origin DNA sequencePrimasome dnaB helicase (unwinds DNA at origin) dnaC binds dnaB dnaG primase (synthesizes RNA primer)DNA gyrase introduces negative supercoils ahead

of the replication forkRep protein helicase (unwinds DNA at fork)SSB binds to single-stranded DNADNA pol III primary replicating polymeraseDNA pol I removes primer and fills gapDNA ligase seals gap by forming 3’, 5’-phosphodiester bond

Properties of DNA polymerases

DNA polymerases of E. coli_

pol I pol II pol III (core)Polymerization: 5’ to 3’ yes yes yesProofreading exonuclease: 3’ to 5’ yes yes yesRepair exonuclease: 5’ to 3’ yes no no

DNA polymerase III is the main replicating enzymeDNA polymerase I has a role in replication to fill gaps and excise primers on the lagging strand, and it is also a repair enzyme and is used in making recombinant DNA molecules

• all DNA polymerases require a primer with a free 3’ OH group• all DNA polymerases catalyze chain growth in a 5’ to 3’ direction• some DNA polymerases have a 3’ to 5’ proofreading activity

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-A to C-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

Mechanisms of Repair

• Mutations that occur during DNA replication are repaired whenpossible by proofreading by the DNA polymerases

• Mutations that are not repaired by proofreading are repairedby mismatch (post-replication) repair followed byexcision repair

• Mutations that occur spontaneously any time are repaired byexcision repair (base excision or nucleotide excision)

Mismatch (post-replication) repair(reduces DNA replication errors 1,000-fold)

5’3’

CH3

CH3

CH3

CH3

• the parental DNA strands are methylated on certain adenine bases

• mutations on the newly replicated strand are identified by scanning for mismatches prior to methylation of the newly replicated DNA

• the mutations are repaired by excision repair mechanisms• after repair, the newly replicated strand is methylated

Excision repair

ATGCUGCATTGATAGTACGGCGTAACTATC

thymine dimer

AT AGTACGGCGTAACTATC

ATGCCGCATTGATAGTACGGCGTAACTATC

ATGCCGCATTGATAGTACGGCGTAACTATC

excinuclease

DNA polymerase

DNA ligase

(~30 nucleotides)

ATGCUGCATTGATACGGCGTAACT

ATGC GCATTGATACGGCGTAACT

AT GCATTGATACGGCGTAACT

deamination

ATGCCGCATTGATACGGCGTAACT

ATGCCGCATTGATACGGCGTAACT

uracil DNA glycosylase

repair nucleases

DNA polymerase

DNA ligase

Base excision repair Nucleotide excision repair

Deamination of cytosine can be repaired

More than 30% of all single base changes that have been detected as a cause of genetic disease have occurred at 5’-mCpG-3’ sites

Deamination of 5-methylcytosine cannot be repaired

cytosine uracil

thymine5’-methyl-cytosine

DNA repair activity

Life

spa

n

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