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G0 G1
phase
Quiescent cells
G2
phase Growth and
M preparation for
phase
cell division
Mitosis
DNA replication is semi-conservative
5’ 3’
3’ 5’
bidirectional replication
replication bubble
fusion of bubbles
5’ 3’
3’ 5’
daughter chromosomes
5’ 3’
3’ 5’
Initiation of DNA synthesis at the E. coli origin (ori)
origin DNA sequence
5’ 3’
3’ A A A 5’
binding of dnaA proteins
A
A A
DNA melting induced
A A A by the dnaA proteins
A
A A G
A A B C
A
dnaB further unwinds the helix
and displaces dnaA proteins
A
A G
A B C RNA primer
A
A
Primasome
G dna B (helicase)
B C dna C
dna G (primase)
template strand
5’ 3’
3’ OH 5’
RNA primer
(~5 nucleotides)
DNA polymerase
5’ 3’
5’
RNA primer
3’
5’
newly synthesized DNA
Discontinuous synthesis of DNA
5’ 3’
5’ 3’ 5’ 3’
3’ 5’ 3’ 5’
3’ 5’
5’
3’
...has to be discontinuous.
3’
5’
direction of lagging strand synthesis
Strand separation at the replication fork causes positive
supercoiling of the downstream double helix
3’
5’
5’
3’
3’
• DNA gyrase is a topoisomerase II, which 5’
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)
Movement of the replication fork
5’
3’ 5’
3’
Movement of the replication fork
5’ RNA primer
Okazaki fragment
RNA primer
RNA primer pol III
5’ 5’
3’
5’ 5’
3’
5’
3’ G Primasome DNA ligase
C B
Single-strand
binding protein
(SSB)
pol III
Achondroplasia 6 to 40
Aniridia 2.5 to 5
Duchenne muscular dystrophy 43 to 105
Hemophilia A 32 to 57
Hemophilia B 2 to 3
Neurofibromatosis -1 44 to 100
Polycystic kidney disease 60 to 120
Retinoblastoma 5 to 12
deletion insertion
CATCACCTGTACCA CATGTCACCTGTACCA
GTAGTGGACATGGT GTACAGTGGACATGGT
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
Cytosine
C G C G
• replication of C-G should give daughter strands each with C-G
C G C A
• tautomer formation C during replication will result in mispairing
and insertion of an improper A in one of the daughter strands
C A 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
cytosine uracil
5’-methyl- thymine
cytosine
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
Correlation between DNA repair
activity in fibroblast cells from
various mammalian species and
the life span of the organism
100
human
elephant
cow
Life span
10
hamster
rat
mouse
shrew
1
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 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