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