dna replication, mutation, and repair
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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
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