3.

DNA Replication, Mutation, Repair

a). DNA replication i). Cell cycle/ semi-conservative replication ii). Initiation of DNA replication iii). Discontinuous DNA synthesis iv). Components of the replication apparatus b). Mutation i). Types and rates of mutation ii). Spontaneous mutations in DNA replication iii). Lesions caused by mutagens c). DNA repair i). Types of lesions that require repair ii). Mechanisms of repair Proofreading by DNA polymerase Mismatched repair Excision repair iii). Defects in DNA repair or replication

The mammalian cell cycle

Rapid growth and preparation for DNA synthesis DNA synthesis and histone synthesis phase

G0
Quiescent cells

G1
phase

phase

phase

G2

Growth and preparation for cell division

Mitosis

DNA replication is semi-conservative

Parental DNA strands

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

Daughter DNA strands

Origins of DNA replication on mammalian chromosomes

origins of DNA replication (every ~150 kb) 5 3 3 5

bidirectional replication

replication bubble fusion of bubbles

5 3
5 3

daughter chromosomes

3 5
3 5

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

origin DNA sequence

5 3

3 5

binding of dnaA proteins

A A A

A
A

DNA melting induced by the dnaA proteins

dnaA proteins coalesce

dnaB and dnaC proteins bind to the single-stranded DNA

A
A A

A
A

B C

dnaB further unwinds the helix

dnaG (primase) binds...

A A A

B C
A

dnaB further unwinds the helix and displaces dnaA proteins

A A A A A A

...and synthesizes an RNA primer

G
RNA primer

B C

B C

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

template strand 3 OH 5 RNA primer (~5 nucleotides)

DNA polymerase
5 5 RNA primer 3

5 newly synthesized DNA

DNA

DNA

Reaction catalyzed by DNA polymerase

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

Discontinuous synthesis of DNA

5 3

3 5

5 3

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.

Each replication fork has a leading and a lagging strand

leading strand (synthesized continuously)

replication fork 5 3 3 5

replication fork

5 3
3 5

5 3

3 5

lagging strand (synthesized discontinuously) 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)

RNA primer direction of leading strand synthesis 3 5 replication fork 5 3 3 5

direction of lagging strand synthesis

Movement of the replication fork

5 3

5 3

Movement of the replication fork

RNA primer Okazaki fragment

RNA primer

RNA primer 5

pol III 5 DNA polymerase III initiates at the primer and elongates DNA up to the next RNA primer

newly synthesized DNA (100-1000 bases) (Okazaki fragment) 5

pol I

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

newly synthesized DNA (Okazaki fragment)

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

Proteins at the replication fork in E. coli

Rep protein (helicase)
pol III 3 5

5 3

G
Single-strand binding protein (SSB)

Primasome

DNA ligase

C B

pol III

DNA gyrase - this is a topoisomerase II, which breaks and reseals the DNA to introduce negative supercoils ahead of the fork

pol I

Components of the replication apparatus

dnaA Primasome dnaB dnaC dnaG DNA gyrase binds to origin DNA sequence helicase (unwinds DNA at origin) binds dnaB primase (synthesizes RNA primer) introduces negative supercoils ahead of the replication fork helicase (unwinds DNA at fork) binds to single-stranded DNA primary replicating polymerase removes primer and fills gap seals gap by forming 3, 5-phosphodiester bond

Rep protein SSB DNA pol III DNA pol I DNA ligase

Properties of DNA polymerases

DNA polymerases of E. coli_
Polymerization: 5 to 3 Proofreading exonuclease: 3 to 5 Repair exonuclease: 5 to 3 pol I pol II pol III (core) yes yes yes yes yes yes yes no no

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

Properties of DNA polymerases a

DNA polymerases of humans

Location nucl Replication yes Repair no Functions 5 to 3 polymerase yes 3 to 5 exonuclease no 5 to 3 exonuclease1 no Primase yes Associates with PCNA2 no Processivity low Strand synthesis lagging
1 activity

nucl no yes yes no no no no

mito yes no yes yes no no no

nucl yes no

nucl (no) yes3

yes yes yes yes no no no no yes no high repair both leading repair

present in associated proteins 2 Proliferating Cell Nuclear Antigen 3involved in transcription-linked DNA repair

Proteins at the replication fork in humans

helicase

leading strand PCNA pol d

3 5

5 3

DNA ligase

SSB

5 to 3 exo associated with the complex

pol e

pol a

topoisomerases I and II

primase activity lagging strand associated with pol a

Mutation

Types and rates of mutation

Type Genome mutation Mechanism chromosome missegregation (e.g., aneuploidy) chromosome rearrangement (e.g., translocation) base pair mutation (e.g., point mutation, or small deletion or insertion Frequency________ 10-2 per cell division

Chromosome mutation

6 X 10-4 per cell division

Gene mutation

10-10 per base pair per cell division or 10-5 - 10-6 per locus per generation

Mutation rates* of selected genes

Gene New mutations per 106 gametes 6 2.5 43 32 2 44 60 5 to 40 to 5 to 105 to 57 to 3 to 100 to 120 to 12

Achondroplasia Aniridia Duchenne muscular dystrophy Hemophilia A Hemophilia B Neurofibromatosis -1 Polycystic kidney disease Retinoblastoma

*mutation rates (mutations / locus / generation) can vary from 10-4 to 10-7 depending on gene size and whether there are hot spots for mutation (the frequency at most loci is 10-5 to 10-6).

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 New germline mutations

each sperm contains ~100 new mutations a normal ejaculate has ~100 million sperm 100 X 100 million = 10 billion new mutations ~1 in 10 sperm carries a new deleterious mutation at a rate of production of ~8 X 107 sperm per day, a male will produce a sperm with a new mutation in the Duchenne muscular dystrophy gene approximately every 10 seconds.

Types of base pair mutations

CATTCACCTGTACCA GTAAGTGGACATGGT

normal sequence

CATCCACCTGTACCA GTAGGTGGACATGGT

transition (T-A to C-G)

CATGCACCTGTACCA GTACGTGGACATGGT

transversion (T-A to G-C)

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

CATCACCTGTACCA GTAGTGGACATGGT

deletion

CATGTCACCTGTACCA GTACAGTGGACATGGT

insertion

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

Tautomeric forms of the DNA bases

Guanine

Thymine

KETO

ENOL

Mutation caused by tautomer of cytosine

Cytosine

Normal tautomeric form

Guanine

Cytosine

Rare imino tautomeric form

Adenine

cytosine mispairs with adenine resulting in a transition mutation

Mutation is perpetuated by replication

C G
C G

C G
C A C A

and

C G
C G

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

and

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

T A

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

Chemical mutagens
Deamination by nitrous acid

Derivation by hydroxylamine

Alkylation by dimethyl sulfate causes depurination

The formation of a quarternary nitrogen destabilizes the deoxyriboside bond and the base is released from deoxyribose

Attack by oxygen radicals

Thymine dimer formation by UV light

Summary of DNA lesions

Missing base Altered base Incorrect base Acid and heat depurination (~104 purines per day per cell in humans) Ionizing radiation; alkylating agents Spontaneous deaminations cytosine to uracil adenine to hypoxanthine

Deletion-insertion
Dimer formation Strand breaks Interstrand cross-links

Intercalating reagents (acridines)

UV irradiation Ionizing radiation; chemicals (bleomycin) Psoralen derivatives; mitomycin C

(Tautomer formation Spontaneous and transient)

Mechanisms of Repair
Mutations that occur during DNA replication are repaired when possible by proofreading by the DNA polymerases

Mutations that are not repaired by proofreading are repaired by mismatched (post-replication) repair followed by excision repair Mutations that occur spontaneously any time are repaired by excision repair (base excision or nucleotide excision)

Mismatched (post-replication) repair

the parental DNA strands are methylated on certain adenine bases
CH3

CH3

5 3
CH3

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

CH3

Excision repair (base or nucleotide)

deamination

ATGCUGCATTGA TACGGCGTAACT
uracil DNA glycosylase thymine dimer

ATGC GCATTGA TACGGCGTAACT

repair nucleases

ATGCUGCATTGATAG TACGGCGTAACTATC
excinuclease

AT GCATTGA TACGGCGTAACT
DNA polymerase b

AT (~30 nucleotides) AG TACGGCGTAACTATC

DNA polymerase b

ATGCCGCATTGA TACGGCGTAACT
DNA ligase

ATGCCGCATTGATAG TACGGCGTAACTATC
DNA ligase

ATGCCGCATTGA TACGGCGTAACT
Base excision repair

ATGCCGCATTGATAG TACGGCGTAACTATC
Nucleotide excision repair

Deamination of cytosine can be repaired

Deamination of 5-methylcytosine cannot be repaired

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

Defects in DNA repair or replication Xeroderma pigmentosum Ataxia telangiectasia Fanconi anemia Bloom syndrome 100 human Cockayne syndrome elephant cow Life span
10

Correlation between DNA repair activity in fibroblast cells from various mammalian species and the life span of the organism 1

hamster rat mouse shrew DNA repair activity

Defects in DNA repair or replication

All are associated with a high frequency of chromosome and gene (base pair) mutations; most are also associated with a predisposition to cancer, particularly leukemia
Xeroderma pigmentosum caused by mutations in genes involved in nucleotide excision repair associated with a 2000-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 increased risk of X-ray 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 Werners syndrome caused by mutations in a DNA helicase gene premature aging