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DNA replication-copying the genetic blueprint

DNA replication-copying the genetic blueprint Marc S. Wold Medical Biochemistry September 13, 2010 Readings: Lippencot -

Marc S. Wold Medical Biochemistry September 13, 2010

Readings:

Lippencot - Chapter 29 Lehninger - Chapter 24

Outline

DNA replication

General mechanism of DNA replication

Replication enzymes

Prokaryotic replication

Eukaryotic replication

Study Questions and Key Concepts

1.

Define stages of replication & semi-discontinuous synthesis

2.

What are the two classes of DNA polymerases? What is the

3.

general mechanism and requirements for DNA polymerase function including the function of DNA polymerase accessory complexes? What are the 7 types of proteins needed for needed for

4.

progression of replication fork? The function of each class at replication fork? What are the similarities and differences between

5.

prokaryotic and eukaryotic DNA replication? Why is a linear chromosome a problem in replication? How

6.

do mammalian cells overcome this problem? What is the enzymatic activity of telomerase and what is its

7.

role in the cell? What is the general mechanism of initiation of replication?

8.

What is the arrangement of origins of replication on eukaryotic chromosomes?

Cellular Information Flow

DNA metabolism

Replication

Transcription

RNA

Translation

DNA

DNA

 
Proteins

Proteins

 

RNA processing

DNA   Proteins   RNA processing DNA Repair Protein degradation Chromatin Structure -

DNA Repair

Protein degradation

Chromatin Structure - organization of DNA in Cell

Cell Cycle regulation - coordination of cellular information flow with other cellular and tissue processes

General mechanism of DNA replication

Nucleotide sequence stores genetic information. • Each strand of a DNA double helix contains all the genetic information. • DNA strands are antiparallel • Cells must accurately duplicate and maintain DNA sequence to ensure genetic integrity

strands are antiparallel •   Cells must accurately duplicate and maintain DNA sequence to ensure genetic
1. DNA Polymerase •   DNA is synthesized by enzymes called DNA polymerases •  

1. DNA Polymerase

DNA is synthesized by enzymes called DNA polymerases • Reaction: primer + dNTP primer-dNMP and PPi Universal properties of DNA polymerases:

Synthesize DNA in a 5’ to 3’ direction

Required an existing DNA strand to copy; require at template

Require a primer; add to a 3’ OH

DNA Replication - definitions

Origin of Replication - DNA sequence at which replication initiates Replication forks - points of replication that move through DNA Unidirectional or bidirectional replication - replication occurs with either 1 or 2 forks, bidirectional replication most common

or bidirectional replication - replication occurs with either 1 or 2 forks, bidirectional replication most common
or bidirectional replication - replication occurs with either 1 or 2 forks, bidirectional replication most common

Semi-discontinuous DNA replication

Two strands are antiparallel & DNA polymerase synthesizes 5’=>3’

At a replication fork the two strands in the double helix must be

synthesized by a different mechanisms at the same time. DNA synthesis is continuous on the leading strand and discontinuous on

the lagging strand. The short DNA fragments on lagging strand are called Okazaki fragments.

DNA polymerase requires a primers so each Okazaki fragment must begin with a primer.

Okazaki fragments . •   DNA polymerase requires a primers so each Okazaki fragment must begin

Definitions: Stages of DNA Replication

Initiation of Replication - The process that leads primer synthesis and assembly of replication fork.

Elongation - The process of DNA synthesis; replication forks move through DNA synthesizing new DNA strands.

Termination - When replication stops. Usually occurs when replication forks meet.

new DNA strands. •   Termination - When replication stops. Usually occurs when replication forks meet.

DNA polymerases

There are two classes of DNA polymerases in cells Classical DNA polymerases (Replicative DNA polymerases) - involved in DNA replication of undamaged DNA and some DNA repair processes Translesion DNA polymerases - involved in DNA repair and DNA synthesis of damaged DNA

Clinical Correlation: Many antiviral agents target viral DNA polymerases. Acyclovir (Acycloguanosine, Zovirax®) - inhibitor of Herpes DNA polymerases Entecavir (Baraclude®) - inhibites hepatitis B virus (HBV) DNA polymerase

DNA polymerases in E. coli –   DNA polymerase and exonuclease activities –   Number
DNA polymerases in E. coli
–   DNA polymerase and exonuclease activities
–   Number of copies per cell and activity vary (e.g. processivity)
– Primary role - replication (III) and repair processes (I & II).
Table for illustrative purpose

There are five DNA polymerases in E. coli - 3 classical & 2 translesion • Classical polymerases:

•   There are five DNA polymerases in E. coli - 3 classical & 2 translesion
DNA polymerase III: a replication machine •   Key concept: DNA Polymerase III holoenzyme is

DNA polymerase III: a replication machine

•   Key concept: DNA Polymerase III holoenzyme is composed of three subcomplexes Table for
•  
Key concept: DNA Polymerase
III holoenzyme is composed of
three subcomplexes
Table for illustrative purpose

DNA Polymerase III holoenzyme is a complex replication machine:

10 subunits, rapid rate of synthesis (1000 nt/sec), accurate (1 error/ 10 5 nt) and high processivity (>10 5 nt before dissociation).

Polymerase accessory factors: Sliding clamps & Clamp loaders

DNA polymerase III achieves

high processivity by being topologically linked to DNA. Sliding clamp: toroid shaped

Beta complex is a sliding clamp. Interacts with core polymerase and holds it on the DNA. Clamp loader: beta is loaded

onto double-stranded DNA by the gamma complex.

A similar mechanism is used other processive DNA polymerases

double-stranded DNA by the gamma complex. •   A similar mechanism is used other processive DNA
double-stranded DNA by the gamma complex. •   A similar mechanism is used other processive DNA

2. Primases - starting nascent DNA chains

Primases synthesize short RNA oligonucleotide primers • Can initiate synthesis on ssDNA de novo (no 3’=OH needed). • Most primases start synthesis at a random sites • Usually part of protein complex at replication fork

•   Most primases start synthesis at a random sites •   Usually part of protein

3. DNA helicases - separation of the double helix

DNA helicases utilize energy of ATP

hydrolysis to disrupt the double helix.

Helicases move along ssDNA in one

direction disrupting hydrogen bonds. (Helicases either move 5’ to 3’ or 3’ to 5’.) Nomenclature - direction of helicase

movement is defined on the strand the helicase binds. (At right is a 5’ to 3’ helicase) Helicases are necessary for movement of a replication fork; usually interact with primase.

is a 5’ to 3’ helicase) Helicases are necessary for movement of a replication fork; usually

4. ssDNA binding proteins (SSBs)

4. ssDNA binding proteins (SSBs) •   SSBs bind ssDNA. –   SSBs prevent formation of

SSBs bind ssDNA.

SSBs prevent formation of secondary structure in ssDNA

SSBs usually interact with other replication proteins; these interactions promote efficient replication

5. DNA nucleases Nucleases DNA or RNA

Nucleases can degrade

  Nucleases DNA or RNA   Nucleases can degrade – DNA from its end 5’ 

DNA from its end 5’ 3’ or

3’ 5’ (exonucleases) or internally (endonuceases)

5’ 3’ exonuclease required in replication to remove RNA primers to give continuous DNA strand

6. DNA ligases

RNA primers to give continuous DNA strand 6. DNA ligases •   DNA ligases form phosphodiester

DNA ligases form phosphodiester bonds; join strands of DNA

Require high energy cofactor (ATP or NAD)

7. Topoisomerases •   Parental strands start replication as a single helix but end replication
7. Topoisomerases •   Parental strands start replication as a single helix but end replication

7. Topoisomerases

Parental strands start replication as a single helix but end replication as one strand of each of two separate helices Topoisomerases release the links between the parental DNA strands

one strand of each of two separate helices •   Topoisomerases release the links between the

Type I Topoisomerases

Type I Topoisomerases •   Type I topoisomerases make a transient ssDNA break that then allow

Type I topoisomerases make a transient ssDNA break that then allow the DNA to swivel • Change links between strands (L) in units of one

Clinical Correlation:

Topoisomerase inhibitors (antibiotics) - quinolones, fluroquinolones (e.g. nalaidixic acid, coumermycin A, and novobiocin).

Topoisomerase I inhibitors (chemotherapy) - camptothecin & less toxic varients: Topotecan (Hycamtin®), irinotecan (Camptosar®) are used in chemotherapy.

Type II Topoisomerases

Type II topoisomerase cause a transient dsDNA break. Change links between strands

(L)

in units of two

Can link or unlink two molecules of DNA (catenation or decatenation).

Form a covalent intermediate with the transiently broken end

(s)

of the DNA.

Most topoisomerases relax supercoiled DNA.

Clinical Correlation:

Topoisomerase II inhibitors - Etoposide phosphate (Eposin®, Etopophos®, VP-16®) - used in chemotherapy.

•   Topoisomerase II inhibitors - Etoposide phosphate (Eposin®, Etopophos®, VP-16®) - used in chemotherapy.

Replication fork and elongation

Table for illustrative purpose
Table for illustrative purpose

Replication proteins act coordinately at the replication fork to promote efficient DNA synthesis • Leading and lagging strand synthesis are coordinated • Movie showing E. coli fork at:

http://www.freesciencelectures.com/video/dna-replication-process/

Replication

Fork

Helicase separating DNA

strands Primase synthesizing

primers for lagging strand Primosome: “helicase/

primase complex” synthesizes multiple primers on the lagging strand SSB stabilizing ssDNA

Topoisomerase

removing links between parental DNA Nuclease removing

primers Processing of Okazaki fragments

• removing links between parental DNA Nuclease removing •   primers Processing of Okazaki fragments
Processing of Okazaki fragments •   Processing of okazaki fragments requires: –   nuclease to

Processing of Okazaki fragments

Processing of okazaki fragments requires:

nuclease to remove primer,

DNA polymerase to fill in gap and

ligase to seal nick

In E. coli, processing requires

DNA pol I (5’ 3’ exonuclease and DNA polymerase activities) and

ligase

Eukaryotic replication: Different proteins, similar functions and mechanism

Table for illustrative Purpose only
Table for
illustrative
Purpose
only

Replication proteins have have conserved functions.

The structures of Pol III beta subunit and PCNA shown a right Clinical Correlation:

PCNA antibody - used in diagnosis as a marker for proliferating cells.

Eukaryotic DNA polymerases

There are at least 15 polymerases in eukaryotic cells • Designated by Greek letters Classical DNA polymerases (Replicative polymerases)

Alpha polymerase & primase - initiation of replication & okazaki fragments

Beta-DNA repair

Gamma - mitochondrial DNA replication

Delta & epsilon - chromosomal DNA replication

Translesion DNA polymerases (DNA repair & DNA synthesis of damaged DNA)

Zeta, eta, theta, … - DNA repair and bypass synthesis

DNA polymerase alpha, delta and epsilon all function at the eukaryotic replication fork

Chromosomal DNA !  E.coli cell Prokaryotes Prokaryotic vs Eukaryotic Replication Human metaphase chromosomes 
Chromosomal DNA !  E.coli cell Prokaryotes Prokaryotic vs Eukaryotic Replication Human metaphase chromosomes 

Chromosomal DNA !

E.coli cell

Prokaryotes

Prokaryotic vs Eukaryotic Replication

Human metaphase chromosomes

Eukaryotes

Replication Human metaphase chromosomes  Eukaryotes differences differences – 20-30 minute cell doubling

differences

differences

20-30 minute cell doubling

Small genomes (10 5 -10 7 )

Single chromosome

Single origin of replication

8 hour S phase

Large genomes (10 7 -10 12 )

Multiple chromosomes

Multiple origins of replication

similarities

similarities

Efficient DNA synthesis

Highly regulated

Efficient DNA synthesis

Highly regulated

Replicate once and only once

Replicate once and only once per

per cell cycle

cell cycle

Coordinate with nutritional

Coordinate replication with cell

state

cycle

DNA damage and checkpoints can stop replication

DNA damage and checkpoints can stop replication

Eukaryotic chromosomes

Eukaryotic chromosomes   • •   Centromere = attachment point for proteins that link the chromosome

Centromere = attachment point for proteins that link the chromosome to the mitotic spindle Telomeres = sequences at the end that help to stabilize the chromosome.

Consequences of linear chromosomes:

DNA ends

Check point activation (ends must not activate checkpoints) 5’ end problem

Telomeres

Telomeres: Specialized

(CCCTAA) n )

structure at the ends of eukarkyotic chromosomes Telomeres contain thousands

of copies of short 6-8 nt, GC- rich repeats. (In humans:

Telomere sequences bound by multiple telomeric proteins and form a specialized, looped nucleoprotein complex (T-loop)

Telomere structure prevents DNA end from being recognized as double strand break

DNA end from being recognized as double strand break Top: Proposed stucture of Telomere loop (T
DNA end from being recognized as double strand break Top: Proposed stucture of Telomere loop (T

Top: Proposed stucture of Telomere loop (T loop)

Bottom: electron micrograph of Mouse telomere

Lagging strand!
Lagging strand!

The problem with 5’ ends of linear genomes)

How to synthesize the 5’ end of the lagging strand.

The last primer must be removed and filled in or else there will be loss of genetic material.

Telomere Synthesis • 3’ (parental ) strand is extended by specialized RNA protein complex: •

Telomere

Synthesis

3’ (parental ) strand is extended by specialized RNA protein complex:

telomerase Telomerase contains a “reverse transcriptase” activity that uses internal

RNA as template to synthesize telomere repeats de novo 5’ strand is synthesized by lagging strand mechanism

Telomerase extension prevents loss of DNA at the 5’ end

Telomerase, cancer, …

Telomerase, cancer, … Aubert & Lansdorp (2008) Physiol Rev 88, 557 Harley et al (1990) Nature

Aubert & Lansdorp (2008) Physiol Rev 88, 557

cancer, … Aubert & Lansdorp (2008) Physiol Rev 88, 557 Harley et al (1990) Nature 354,

Harley et al (1990) Nature 354, 458

Physiol Rev 88, 557 Harley et al (1990) Nature 354, 458 “Accelerated telomere shortening in response

“Accelerated telomere shortening in response to life stress”!

Epel et al (2004) PNAS 101, 17312 !

Most human cells do not express telomerase and telomere length

decreases as normal cells grow. Immortalized cells usually have induced expression of telomerase

Normal cells that have telomerase expression activated do not stop

growing. Telomerase expression is a critical factor in transformation of cells and the

cancer development. Telomere length decreases with age (and stress).

General Mechanism of Initiation

Specific complexes form at origin • Origin is activated causing local opening of the DNA helix and loading of DNA helicase • Primase binds and synthesizes initial RNA primers • DNA polymerase extends primers. Leading and lagging strand synthesis established • The primary (only) specificity in DNA replication is at initiation.

and lagging strand synthesis established •   The primary (only) specificity in DNA replication is at
Chromosomal DNA !  E.coli cell Prokaryotes Prokaryotic vs Eukaryotic Replication Human metaphase chromosomes 
Chromosomal DNA !  E.coli cell Prokaryotes Prokaryotic vs Eukaryotic Replication Human metaphase chromosomes 

Chromosomal DNA !

E.coli cell

Prokaryotes

Prokaryotic vs Eukaryotic Replication

Human metaphase chromosomes

Eukaryotes

Replication Human metaphase chromosomes  Eukaryotes differences differences – 20-30 minute cell doubling

differences

differences

20-30 minute cell doubling

Small genomes (10 5 -10 7 )

Single chromosome

Single origin of replication

8 hour S phase

Large genomes (10 7 -10 12 )

Multiple chromosomes

Multiple origins of replication

similarities

similarities

Efficient DNA synthesis

Highly regulated

Efficient DNA synthesis

Highly regulated

Replicate once and only once

Replicate once and only once per

per cell cycle

cell cycle

Coordinate with nutritional

Coordinate replication with cell

state

cycle

DNA damage and checkpoints can stop replication

DNA damage and checkpoints can stop replication

Eukaryotic origins of replication

Eukaryotes have long linear chromosomes

Initiation must be tightly coordinated (once and only once)

Each chromosome has multiple origins (~1 origin every 100 kbp)

chromosome has multiple origins (~1 origin every 100 kbp) In humans: • There is not a

In humans:

There is not a defined

origin sequence. Proteins required for

initiation are known.

Origin recognition complex (ORC): DNA binding protein required for initiation of replication.

Eukaryotic

initiation

Replication is “licenced” in M/G1. The pre-replicative complex (required for initiation) forms on DNA but is not active. • Helicase is activated by S phase cdk (& other kinases active in S) • Some initiation proteins are degraded after replication starts • Only some proteins shown

proteins are degraded after replication starts •   Only some proteins shown Table for illustrative purpose

Table for illustrative purpose

General Mechanism of DNA Replication

General Mechanism of DNA Replication • Initiation - specific complexes form at origin • Origin is

Initiation - specific complexes form at origin

Origin is activated causing local opening of the

DNA helix and loading of DNA helicase Primase binds and synthesizes initial RNA

primers DNA polymerase extends primers. Leading and lagging strand synthesis established

initial RNA •   primers DNA polymerase extends primers. Leading and lagging strand synthesis established

DNA mutagenesis and repair

99:163 - Medical Biochemistry September 15, 2010

Marc S. Wold

Readings:

Lippencott - Chapter 29 Lehninger - Chapter 25

September 15, 2010 Marc S. Wold Readings: Lippencott - Chapter 29 Lehninger - Chapter 25 Hakem

Hakem (2008) EMBO J. 27, 589 !

DNA repair

Outline

DNA repair proteins and mutations

Types of damage

Cellular DNA Repair pathways

Defects in DNA metabolism and human disease

Study Questions & Key Concepts

1.

What are the types of modifications cause DNA

proofreading, mismatch repair, NER, BER, error-prone

2.

damage? How is damaged DNA changed to a genetic mutation?

3.

What DNA repair pathways/processes occur prior to

4.

DNA replication? During or after DNA replication? What is the general mechanism of (what type of damage

5.

is repaired and what is the general mechanism of:

repair (translesion synthesis), NHEJ, recombinational repair? What pathway is defective in Xeroderma pigmentosum?

6.

HNPCC? Fanconi’s Anemia? How do the two pathways for repairing double-stranded DNA breaks differ?

Cellular Information Flow

DNA metabolism

Replication

Transcription

RNA processing

Flow DNA metabolism Replication Transcription RNA processing RNA Translation DNA Proteins Protein degradation DNA Repair

RNA

Translation

Replication Transcription RNA processing RNA Translation DNA Proteins Protein degradation DNA Repair Chromatin
Replication Transcription RNA processing RNA Translation DNA Proteins Protein degradation DNA Repair Chromatin

DNA

Proteins

Protein degradation

DNA Repair

Chromatin Structure - organization of DNA in Cell

Cell Cycle regulation - coordination of cellular information flow with other cellular and tissue processes

Dangerous environments

Dangerous environments •   Many environments are dangerous •   Danger can be mitigated but not

Many environments are dangerous • Danger can be mitigated but not always eliminated

This is also true in cells where the environment contributes to DNA damage

Dangerous environments

Many environments are dangerous • Danger can be mitigated but not always eliminated

This is also true in cells where the environment contributes to DNA damage

in cells where the environment contributes to DNA damage •   Environmental chemicals •   Radiation

Environmental chemicals • Radiation • Natural chemistry • Normal metabolic processes

Environmental Chemicals

Many chemicals can modify DNA.

Aflatoxin (peanut butter)

Cyclic hydrocarbons (grilling

and cooking)

Nitrates - Sodium nitrate

growth of Clostridium

(preservative in prepared meat products-prevents

botulinum. Converted to nitrosamines by in stomach) Reactive oxygen species (O 2 - , HO•) Produced by phagocytes (to kill pathogens), during electron transport in mitochondria metabolism and by oxidizing agents (H 2 O 2 )

metabolism and by oxidizing agents (H 2 O 2 ) •   Most modified bases are

Most modified bases are repaired by one or more repair systems.

Types of DNA damage Depurination (G or A) Spontaneous Single strand break Caused by reactive
Types of DNA damage
Depurination (G or A)
Spontaneous
Single strand break
Caused by reactive
chemicals, oxidative
damage, radiation
Thymine dimer (& other
intra-base bonds)
Caused by UV
Deamination (C, A, G)
Spontaneous
U !
inter-strand Crosslinks
Caused by reactive
chemicals, radiation, free
radicals
Intercalation (binding of
chemicals between base pairs)
Cause by chemical exposure
*
Chemical modification
(alkalyation, oxidative
damage, …)
*
Caused by reactive
chemicals, reactive
oxygen species
Double strand break
Caused by reactive
chemicals, oxidative
damage, radiation
C !
Mismatchs
Caused by replication
errors
Frequency of DNA damage DNA lesions/mammalian cell/day (from J. Bartek) •   50,000 single-strand breaks
Frequency of DNA damage
DNA lesions/mammalian cell/day
(from J. Bartek)
•   50,000 single-strand breaks
•   10,000 depurinations
U !
•  
•  
•  
•  
•  
600 deaminations
2,000 oxidative lesions
5,000 alkylated bases
10 inter-strand crosslinks
10 double-strand breaks
*
*
C !

DNA repair:

Why does DNA damage cause mutations?

DNA repair: Why does DNA damage cause mutations? •   Damaged (chemically modified) DNA has altered

Damaged (chemically modified) DNA has altered (or no) base pairing • DNA polymerase can mis- read a damaged template and incorporate the wrong base

Replication converts damage to a mutation •   Making a mutation : during replication, the

Replication converts damage to a mutation

Making a mutation: during replication, the wrong base is inserted opposite a damaged base causing a permanent change in genetic information - a mutation.

Replication fork arrest: the replicative DNA polymerasecannot copy the damaged base causing the replication fork to stall.

Types of DNA Repair

DNA damage occurs as a result of environmental (chemical modification, radiation, etc.) insult, natural cellular processes (reactive oxygen species) or fundamental chemistry. All damage needs to be repaired. !

Replication fidelity and Post replicative repair

Proof-reading

mismatch repair

Damage Repair

(during replication)

Nucleotide Excision

Base Excision

Direct reversal

Transcription Coupled

Specialized repair (inter-strand cross links, specialized damage)

Translesion Synthesis & Error-prone Repair (SOS) • Double-strand Break repair

Nonhomologous End Joining (NHEJ)

Recombinational repair

Types of DNA Repair

DNA damage occurs as a result of environmental (chemical modification, radiation, etc.) insult, natural cellular processes (reactive oxygen species) or fundamental chemistry. All damage needs to be repaired. !

Replication fidelity and Post replicative repair

Proof-reading

mismatch repair

Damage Repair

(during replication)

Nucleotide Excision

Base Excision

Direct reversal

Transcription Coupled

Specialized repair (inter-strand cross links, specialized damage)

Translesion Synthesis & Error-prone Repair (SOS) • Double-strand Break repair

Nonhomologous End Joining (NHEJ)

Recombinational repair

DNA Polymerase • All DNA polymerases: – Synthesize DNA in a 5’ to 3’ direction

DNA Polymerase

All DNA polymerases:

Synthesize DNA in a 5’ to 3’ direction

 

Required an existing DNA strand to copy; require at template

 
 

Require a primer; add to a 3’ OH

 

DNA polymerases can have several intrinsic enzyme activities:

DNA polymerase (all)

3’ to 5’ exonuclease (many)

5’ to 3’ exonuclease (a few - E.coli Pol I)

 

DNA Polymerase accuracy

 
 

Base pairing provides specificity of 100.

Enzyme selectivity 100-1000-fold (active site the shape of a base pair). (Note-only true in classical DNA polymerases.)

Proofreading 3’ 5’ exonucease increases the fidelity of DNA synthesis by 100-fold.

Mismatch repair increases fidelity by 100-fold Total 10 9

Repairs base

mismatches.

 

Mismatch repair - E. coli MutHLS

Mismatch repair - E. coli MutHLS

Uses methyl groups as marker for parent (correct) strand.

Acts after replication but before methylation of nascent strand

A nick is created opposite site of methylation

Intervening DNA between nick and mismatch is removed

Gap is filled by DNA polymerase

Similar pathways exist in eukaryotic cells (uses nicks in nascent strand to direct repair)

DNA metabolism and disease II:

Human mismatch repair Defects in mismatch repair genes lead to human disease www.nitisurgical.com!
Human mismatch repair
Defects in
mismatch
repair
genes lead
to human
disease
www.nitisurgical.com!

Hereditary nonpolyposis colon cancer (HNPCC)

Accounts for 5% of colon cancer

Affected individuals have an 80% chance of developing cancer during their life time

Caused by mutations in one of genes involved in mismatch repair. (e.g hMLH1, hMSH2 or one of other mismatch repair proteins).

Autosomal dominant!

Types of DNA Repair

DNA damage occurs as a result of environmental (chemical modification, radiation, etc.) insult, natural cellular processes (reactive oxygen species) or fundamental chemistry. All damage needs to be repaired. !

Replication fidelity and Post replicative repair

Proof-reading

mismatch repair

Damage Repair

(during replication)

(anytime)

Nucleotide Excision

Base Excision

Direct reversal

Transcription Coupled

Specialized repair (inter-strand cross links, specialized damage)

Translesion Synthesis & Error-prone Repair (SOS) • Double-strand Break repair

Nonhomologous End Joining (NHEJ)

Recombinational repair

Bulky adducts Modifications that disrupt the DNA duplex are called bulky adducts. Caused by: •
Bulky adducts Modifications that disrupt the DNA duplex are called bulky adducts. Caused by: •

Bulky adducts

Modifications that disrupt the DNA duplex are called bulky adducts.

Caused by:

Ultraviolet light • Chemical modifications (alkalytion) • Some types of oxidative damage

UV- causes interbase crosslinks: pyrimidine (cyclobutane) dimers.

Nucleotide Excision Repair

Nucleotide excison repair (NER) is the major pathway for repairing damaged bases (pyrimidine dimers, alkylated DNA and other bulky adducts).

•   •  
•  

NER protein complex (called Excinuclease) binds to site of damage and cuts damaged strand on either side creating a small gap Defects in this pathway result in the disease Xeroderma Pigmentosum

Clinical Correlation

Xeroderma pigmentosum (Groups A-G) Rare genetic disorder that is caused by mutations in proteins involved in NER Patients have thin, unevenly pigmented skin. Also causes spidery blood vessels in the skin (telangiectasia). Very high sensitivity to sunlight and high incidence of cancer. Patients often develop skin cancer before age of five Very rare: ~2/million live births in Western Europe (Kleijer et al 2008, DNA Repair 7,744).

rare: ~2/million live births in Western Europe (Kleijer et al 2008, DNA Repair 7,744). •  

Base Excision Repair

Base excision repair (BER) removes modified or damaged bases including alkylated DNA, oxidative damage, some bulky adducts. Glycosydic bonds linking the damaged base to the DNA are cleaved by specific glycosylases. The Abasic site (deoxyribose with no base attached) is recognized by an AP-endonuclease which nicks the damaged strand.

•   •  
•  

Nuclease & polymerase synthesize across AP site. Nick sealed by DNA ligase.

Why T in DNA but U in RNA?

  •   •   • •   Why T in DNA but U in RNA?

In eukaryotes, cytosine in CpG sequences is methylated. Me- CpG regions are important in gene regulation during development and in imprinting. Deamination of 5-methyl C produces thymine. This is not recognized as damage.

Cytosine undergoes deamination to give uracil. This reaction is spontaneous (>500 hundred per human cell per day) and can be enhanced by oxidizing agents. This causes a change in base pairing. If uracil normally found in DNA, cell could not distinguish deaminated C from normal U and would not repair properly. Uracil in DNA treated as damage and repaired by base excision repair (Uracil N-glycosylase removes U).

Other Repair Processes

Direct reversal - O 6 - methlyguanidine-DNA methyltransferase - This “enzyme” transfers methy or ethyl group from O 6 to Cys. Repairing DNA but inactivating “enzyme”.

O 6 to Cys. Repairing DNA but inactivating “enzyme”. Direct reversal - photolyase- uses light to

Direct reversal - photolyase- uses light to directly reverse light induces pyrimidine dimers. Found in most organisms but not mammals Transcription coupled repair - Transcribed strand is repaired more efficiently than non-transcribed strand in eukaryotes. NER proteins are involved. (Cockaynes Syndrome – symptoms similar to XP but defect is in transcription coupled repair)

Inter-strand cross-links

Repair of cross-linked DNA remains poorly understood.

Repair requires other repair repair proteins (NER, HR) and an additional set of proteins

Fanconi Anemia is caused by a defect in proteins involved in repair of inter-strand cross-links. (Fanconi patients have normal sensitivity to most DNA damaging agents but are very sensitive to cross-linking agents.)

agents but are very sensitive to cross-linking agents.) Dr. Carol Wynman, Erasmus Medical Center ! •

Dr. Carol Wynman, Erasmus Medical Center!

Fanconi Anemia is a form of

aplastic anemia. Patients also develop acute myeloid leukemia (AML) and carcinomas at elevated rates Recessive mutation in one of

~15 genes (includes Fanconi genes and BRCA2). Estimated 1-300 in US is a carrier. Incidence 1~360,000

Clinical correlation Cisplatin (Platinol-AQ®) – widely used in chemotherapy. Forms DNA cross-link.

Types of DNA Repair

DNA damage occurs as a result of environmental (chemical modification, radiation, etc.) insult, natural cellular processes (reactive oxygen species) or fundamental chemistry. All damage needs to be repaired. !

Replication fidelity and Post replicative repair

Proof-reading

mismatch repair

Damage Repair

(during replication)

(anytime)

Nucleotide Excision

Base Excision

Direct reversal

Transcription Coupled

Specialized repair (interstrand cross links, specialized damage)

Tranlesion Synthesis & Error-prone Repair (SOS)

Double-strand Break repair

(during replication)

Nonhomologous End Joining (NHEJ)

Recombinational repair

Error Prone Repair •   •   When the replication fork encounters a unrepaired lesion
Error Prone Repair
•  
When the replication fork encounters a unrepaired lesion or a
strand break, the fork stalls. Specialized repair processes
(translesion synthesis, recombinational repair, fork restart)
allow synthesis to continue.
Pathway requires translesion DNA polymerases.
•  
Translesion
synthesis -
Translesion DNA
polymerases
incorporate
nucleotides
opposite damage
allowing classical
polymerases to
continue
synthesis.
Strand break
Error-prone repair -
Translesion synthesis

Future of Medicine:

Repair vs. mutagenesis

Mutations in translesion polymerases affect mutation rate XP-variant (XP-V) have mutations in DNA polymerase eta (η) and increased rate of mutations. This is caused by cells inability to carry out DNA replication in the presence of DNA damage Could cells be made less sensitive to mutagens by modulating the activities of classical and translesion DNA polymerases?

to mutagens by modulating the activities of classical and translesion DNA polymerases? M.T. Washington, U of

M.T. Washington, U of Iowa!

Types of DNA Repair

DNA damage occurs as a result of environmental (chemical modification, radiation, etc.) insult, natural cellular processes (reactive oxygen species) or fundamental chemistry. All damage needs to be repaired. !

Replication fidelity and Post replicative repair

Proof-reading

mismatch repair

Damage Repair

(during replication)

(anytime)

Nucleotide Excision

Base Excision

Direct reversal

Transcription Coupled

Specialized repair (interstrand cross links, specialized damage)

Tranlesion Synthesis & Error-prone Repair (SOS) Double-strand Break repair

Nonhomologous End Joining (NHEJ)

Recombinational repair

(any time) (after replication)

Double- strand break repair There are two general pathways for repairing ds-DNA breaks:   •

Double-

strand break repair

There are two general pathways for repairing ds-DNA breaks:

Non-homologous end-joining: repair with loss of several bases. (Predominant pathway in mammalian cells) Homologous end-joining or recombination: completely restores sequence from second copy of DNA. (Predominant pathway in bacteria and unicellular eukaryotes.) Both processes involve MRN complex (MRE11, RAD50, NBS1)

Non-

homologous End Joining

Proteins required non- homologous end joining include: end recognition (Ku, DNA-PK ) and processing nucleases (Rad50, MRE11, NBS1, and ligase). Some DNA is lost at the junction

end recognition (Ku, DNA-PK ) and processing nucleases (Rad50, MRE11, NBS1, and ligase). Some DNA is
Recombinational ds-break repair •   Recombinational repair requires resection of the DNA ends and then
Recombinational ds-break repair •   Recombinational repair requires resection of the DNA ends and then

Recombinational ds-break repair

Recombinational repair requires resection of the DNA ends and then general recombination • Note involvement of ATM, NBS1, BRCA and MRE proteins

General

recombination

•   •  
•  

Homologous

recombination

Recombination initiates from either a double-stranded or single-stranded break. Strand invasion leads to the formation of joint molecule - Holliday junction.

Homologous

2 Holliday Junctions
2 Holliday Junctions

recombination

Strand invasion leads to the formation of joint molecule - Holliday junction. Migration and resolution of Holliday junction leads to exchange of DNA sequences. Catalyzed by recA in bacteria, Rad51 in eukaryotes Resolution of Holliday junction results in:

Repaired break

Recombined DNA

DNA metabolism and disease III

NBS !
NBS !

Phenotypes observed

NBS !
NBS !

Genomic instability (ie. Translocations & Telomere shortening)

Increased risk of cancer

Premature aging phenotype (ie. Cataracts, Diabetes & Grey hair)

Developmental defects (ie. Facial defects, Shorter, Infertile)

Neurological defects (ie. Muscle control & Retardation)

Immunological defects (ie. Lung infections, B/T-cell)

Homozygous phenotypes are often lethal

There are wide variations observed in severity of defect(s) (e.g. type and frequency of cancer)

•   Damage activated sensor kinases! •   – RecQ Helicases! MRN complex!
•  
Damage
activated
sensor
kinases!
•  
RecQ
Helicases!
MRN
complex!

DNA metabolism and disease III

Cellular Signaling - Checkpoints

Severe-Combined Immunodeficiency (DNA-PK) Ataxia Telangiectasia (ATM) Seckel Syndrome (ATR) null lethal Cancer (p53, menin)

WS ! Age 14 Age 48 !
WS !
Age 14
Age 48 !

DNA Repair

Recombination

Bloom’s Syndrome (BLM) Werner’s Syndrome (WRN)

Rothmund-Thompson Syndrome (RecQ4)

Familial Breast Cancer (BRCA1 and BRCA2)

AT-Like Disease (Mre11)

Nijmegen Break Syndrome (NBS1) null lethal Fanconi Anemia (FAA genes, BRCA2) null lethal

null lethal

BS !
BS !

Nucleotide Excision Repair

Xeroderma Pigmentosum (groups A-G) (e.g. XPA, XPG proteins)

For reference only-not on exam