Genetics, Chapter 3, DNA Replication Lectures (slides)
-
Upload
ali-hassan-al-qudsi -
Category
Documents
-
view
808 -
download
0
description
Transcript of Genetics, Chapter 3, DNA Replication Lectures (slides)
3 lectures: DNA Replication, Mutation, Repair
Learning Objectives for Lecture 2:
• Understand the general mechanism of DNA replication • Understand the need for a primer for DNA replication • Understand the dynamics of DNA strand synthesis on the leading and lagging
strands of the replication fork • Understand the general functions of the proteins involved in DNA replication
at the replication fork • Understand the role of DNA gyrase in DNA replication and know the class of
antibiotics that inhibits this enzyme • Understand the types of mutations and the rates with which DNA is mutated • Understand the mechanism by which mutations are generated • Understand the basic types of DNA repair and when they take place • Understand the reason for 5'-mCpG mutation hot spots in DNA • Understand the significance of high fidelity DNA replication and for the
presence of DNA repair mechanisms for cellular function and for human disease
2. DNA Replication, Mutation, 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
Section 1
General Concepts of DNA Replication
DNA replication
• A reaction in which daughter DNAs are synthesized using the parental DNAs as the template.
• Transferring the genetic information to the descendant generation with a high fidelity
replication
parental DNAdaughter DNA
Daughter strand synthesis
• Chemical formulation:
• The nature of DNA replication is a series of 3´- 5´phosphodiester bond formation catalyzed by a group of enzymes.
Phosphodiester bond formation
Template: double stranded DNA
Substrate: dNTP
Primer: short RNA fragment with a free 3´-OH end
Enzyme: DNA-dependent DNA polymerase (DDDP),
other enzymes,
protein factor
DNA replication system
Characteristics of replication
Semi-conservative replication
Bidirectional replication
Semi-continuous replication
High fidelity
§1.1 Semi-Conservative Replication
Semiconservative replication
Half of the parental DNA molecule is conserved in each new double helix, paired with a newly synthesized complementary strand. This is called semiconservative replication
Semiconservative replication
Experiment of DNA semiconservative replication
"Heavy" DNA(15N)
grow in 14N medium
The first generation
grow in 14N medium
The second generation
Significance
The genetic information is ensured to be transferred from one generation to the next generation with a high fidelity.
§1.2 Bidirectional Replication
• Replication starts from unwinding the dsDNA at a particular point (called origin), followed by the synthesis on each strand.
• The parental dsDNA and two newly formed dsDNA form a Y-shape structure called replication fork.
3'
5'
5'
3'
5'
3'
5'3'
direction of replication
Replication fork
Bidirectional replication
• Once the dsDNA is opened at the origin, two replication forks are formed spontaneously.
• These two replication forks move in opposite directions as the syntheses continue.
Bidirectional replication
Replication of prokaryotes
The replication process starts from the origin, and proceeds in two opposite directions. It is named replication.
Replication of eukaryotes
• Chromosomes of eukaryotes have multiple origins.
• The space between two adjacent origins is called the replicon, a functional unit of replication.
origins of DNA replication (every ~150 kb)
§1.3 Semi-continuous Replication
The daughter strands on two template strands are synthesized differently since the replication process obeys the principle that DNA is synthesized from the 5´ end to the 3´end.
5'
3'
3'
5'
5'
direction of unwinding3'
On the template having the 3´- end, the daughter strand is synthesized continuously in the 5’-3’ direction. This strand is referred to as the leading strand.
Leading strand
Semi-continuous replication
3'
5'
5'3'
replication direction
Okazaki fragment
3'
5'
leading strand
3'
5'
3'
5'replication fork
• Many DNA fragments are synthesized sequentially on the DNA template strand having the 5´- end. These DNA fragments are called Okazaki fragments. They are 1000 – 2000 nt long for prokaryotes and 100-150 nt long for eukaryotes.
• The daughter strand consisting of Okazaki fragments is called the lagging strand.
Okazaki fragments
Continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand represent a unique feature of DNA replication. It is referred to as the semi-continuous replication.
Semi-continuous replication
Section 2
Enzymology
of DNA Replication
Enzymes and protein factors
protein Mr # function
Dna A protein 50,000 1 recognize origin
Dna B protein 300,000 6 open dsDNA
Dna C protein 29,000 1 assist Dna B binding
DNA pol Elongate the DNA strands
Dna G protein 60,000 1 synthesize RNA primer
SSB 75,600 4 single-strand binding
DNA topoisomerase 400,000 4 release supercoil constraint
• The first DNA- dependent DNA polymerase (short for DNA-pol I) was discovered in 1958 by Arthur Kornberg who received Nobel Prize in physiology or medicine in 1959.
§2.1 DNA Polymerase
DNA-pol of prokaryotes
• Later, DNA-pol II and DNA-pol III were identified in experiments using mutated E.coli cell line.
• All of them possess the following biological activity.
1. 53 polymerizing
2. exonuclease
DNA-pol of E. coli
DNA-pol I
• Mainly responsible for proofreading and filling the gaps, repairing DNA damage
Klenow fragment
• small fragment (323 AA): having 5´→3´ exonuclease activity
• large fragment (604 AA): called Klenow fragment, having DNA polymerization and 3´→5´exonuclease activity
N end C end
caroid
DNA-pol Ⅰ
DNA-pol II
• Temporary functional when DNA-pol I and DNA-pol III are not functional
• Still capable for doing synthesis on the damaged template
• Participating in DNA repairing
DNA-pol III
• A heterodimer enzyme composed of ten different subunits
• Having the highest polymerization activity (105 nt/min)
• The true enzyme responsible for the elongation process
Structure of DNA-pol III
α : has 5´→ 3´ polymerizing activity
ε : has 3´→ 5´ exonuclease activity and plays a key role to ensure the replication fidelity.
θ: maintain heterodimer structure
DNA-pol of eukaryotes
DNA-pol : elongation DNA-pol III
DNA-pol : initiate replication and synthesize primers
DnaG, primase
DNA-pol : replication with low fidelity
DNA-pol : polymerization in mitochondria
DNA-pol : proofreading and filling gap
DNA-pol I
repairing
§2.2 Primase
• Also called DnaG
• Primase is able to synthesize primers using free NTPs as the substrate and the ssDNA as the template.
• Primers are short RNA fragments of a several decades of nucleotides long.
• Primers provide free 3´-OH groups to react with the -P atom of dNTP to form phosphoester bonds.
• Primase, DnaB, DnaC and an origin form a primosome complex at the initiation phase.
§2.3 Helicase
• Also referred to as DnaB.
• It opens the double strand DNA with consuming ATP.
• The opening process with the assistance of DnaA and DnaC
§2.4 SSB protein
• Stand for single strand DNA binding protein
• SSB protein maintains the DNA template in the single strand form in order to
• prevent the dsDNA formation; • protect the vulnerable ssDNA from
nucleases.
§2.5 Topoisomerase
• Opening the dsDNA will create supercoil ahead of replication forks.
• The supercoil constraint needs to be released by topoisomerases.
• The interconversion of topoisomers of dsDNA is catalyzed by a topoisomerase in a three-step process: • Cleavage of one or both strands
of DNA• Passage of a segment of DNA
through this break• Resealing of the DNA break
• Also called -protein in prokaryotes.
• It cuts a phosphoester bond on one DNA strand, rotates the broken DNA freely around the other strand to relax the constraint, and reseals the cut.
Topoisomerase I (topo I)
• It is named gyrase in prokaryotes.
• It cuts phosphoester bonds on both strands of dsDNA, releases the supercoil constraint, and reforms the phosphoester bonds.
• It can change dsDNA into the negative supercoil state with consumption of ATP.
Topoisomerase II (topo II)
3'
5'
5'
3'RNAase
POH
3'
5'
5'
3'
DNA polymerase
P
3'
5'
5'
3'
dNTP
DNA ligase
3'
5'
5'
3'
ATP
§2.6 DNA Ligase
• Connect two adjacent ssDNA strands by joining the 3´-OH of one DNA strand to the 5´-P of another DNA strand.
• Sealing the nick in the process of replication, repairing, recombination, and splicing.
§2.7 Replication Fidelity
• Replication based on the principle of base pairing is crucial to the high accuracy of the genetic information transfer.
• Enzymes use two mechanisms to ensure the replication fidelity.
– Proofreading and real-time correction
– Base selection
• DNA-pol I has the function to correct the mismatched nucleotides.
• It identifies the mismatched nucleotide, removes it using the 3´- 5´ exonuclease activity, add a correct base, and continues the replication.
Proofreading and correction
3´→5´ exonuclease activity excise mismatched
nuleotides
5´→3´ exonuclease activitycut primer or excise mutated segment
C T T C A G G A
G A A G T C C G G C G
5' 3'
3' 5'
Exonuclease functions
Section 3
DNA Replication Process
• Initiation: recognize the starting point, separate dsDNA, primer synthesis, …
• Elongation: add dNTPs to the existing strand, form phosphoester bonds, correct the mismatch bases, extending the DNA strand, …
• Termination: stop the replication
Sequential actions
• The replication starts at a particular point called origin.
• The origin of E. coli, ori C, is at the location of 82.
• The structure of the origin is 248 bp long and AT-rich.
§3.1 Replication of prokaryotes
a. Initiation
Genome of E. coli
• Three 13 bp consensus sequences• Two pairs of anti-consensus repeats
Structure of ori C
Formation of preprimosome
• DnaA recognizes ori C.
• DnaB and DnaC join the DNA-DnaA complex, open the local AT-rich region, and move on the template downstream further to separate enough space.
• DnaA is replaced gradually.
• SSB protein binds the complex to stabilize ssDNA.
Formation of replication fork
• Primase joins and forms a complex called primosome.
• Primase starts the synthesis of primers on the ssDNA template using NTP as the substrates in the 5´- 3´ direction at the expense of ATP.
• The short RNA fragments provide free 3´-OH groups for DNA elongation.
Primer synthesis
• The supercoil constraints are generated ahead of the replication forks.
• Topoisomerase binds to the dsDNA region just before the replication forks to release the supercoil constraint.
• The negatively supercoiled DNA serves as a better template than the positively supercoiled DNA.
Releasing supercoil constraint
Dna ADna B Dna C
DNA topomerase
5'3'
3'
5'
primase
Primosome complex
• dNTPs are continuously connected to the primer or the nascent DNA chain by DNA-pol III.
• The core enzymes ( 、、 and ) catalyze the synthesis of leading and lagging strands, respectively.
• The nature of the chain elongation is the series formation of the phosphodiester bonds.
b. Elongation
• The synthesis direction of the leading strand is the same as that of the replication fork.
• The synthesis direction of the latest Okazaki fragment is also the same as that of the replication fork.
• Primers on Okazaki fragments are digested by RNase.
• The gaps are filled by DNA-pol I in the 5´→3´direction.
• The nick between the 5´end of one fragment and the 3´end of the next fragment is sealed by ligase.
Lagging strand synthesis
3'
5'
5'
3'
RNAase
POH
3'
5'
5'
3'
DNA polymerase
P
3'
5'
5'
3'
dNTP
DNA ligase
3'
5'
5'
3'
ATP
• The replication of E. coli is bidirectional from one origin, and the two replication forks must meet at one point called ter at 32.
• All the primers will be removed, and all the fragments will be connected by DNA-pol I and ligase.
c. Termination
§3.2 Replication of Eukaryotes
• DNA replication is closely related with cell cycle.
• Multiple origins on one chromosome, and replications are activated in a sequential order rather than simultaneously.
Cell cycle
• The eukaryotic origins are shorter than that of E. coli.
• Requires DNA-pol (primase activity) and DNA-pol (polymerase activity and helicase activity).
• Needs topoisomerase and replication factors (RF) to assist.
Initiation
• DNA replication and nucleosome assembling occur simultaneously.
• Overall replication speed is compatible with that of prokaryotes.
b. Elongation
3'
5'
5'
3'
3'
5'
5'
3'
connection of discontinuous
3'
5'
5'
3'
3'
5'
5'
3'
segment
c. Termination
• The terminal structure of eukaryotic DNA of chromosomes is called telomere.
• Telomere is composed of terminal DNA sequence and protein.
• The sequence of typical telomeres is rich in T and G.
• The telomere structure is crucial to keep the termini of chromosomes in the cell from becoming entangled and sticking to each other.
Telomere
• The eukaryotic cells use telomerase to maintain the integrity of DNA telomere.
• The telomerase is composed of
telomerase RNA telomerase association protein telomerase reverse transcriptase
• It is able to synthesize DNA using RNA as the template.
Telomerase
VEDIO….TELOMERASE
• Telomerase may play important roles is cancer cell biology and in cell aging.
Significance of Telomerase
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