DNA REPLICATION and REPAIR

By DR. PHYLIS C. RIO

Learning Objectives

1. State importance of the study of replication2. Trace the flow of genetic information3. Define replication4. Describe the process of DNA synthesis, giving

the roles of the enzymes involved 4.1. Explain and illustrate semiconservative

replication 4.2. Discuss the unwinding of the DNA helix

4.3. Discuss the formation of the two kind strands

5. Discuss the processes involved in the formation of the new DNA chain giving roles of all the enzymes involved

5.1. chain initiation 5.2. chain elongation 5.3. removal of RNA primer and ligation6. Describe the different processes of DNA repair

DNA Functions

• Replication and expression• 1.DNA must be able to replicate itself so that

the information coded into its primary structure is transmitted faithfully to progeny

• 2. The information must be expressed, expression is through RNA intermediates which in turn act as template for synthesis of protein in body

Central Dogma

• Shows relationship of DNA to RNA and to protein

• Indicates three general mechanisms for transfer of information

Replication ( DNA to DNA ) Transcription ( DNA to RNA ) Translation ( RNA to protein )

DNA Replication

• Process by which two new strands of DNA are made using the two existing strands as templates

• Semiconservative – each strand of the parental molecule serves as a template for synthesis of a new strand; resulting daughter DNA molecules consist of one intact template strand ( half the original helix ) and a newly replicated complementary strand

Salient Features of Replication

• Each strand of DNA serves as a template over which a new complementary strand is synthesized

• Base pairing rule is always maintained. New strand is joined to old strand by hydrogen bonds between base pairs (A with T, G with C)

• Polymerization of the new strand of DNA is taking place from 5’ to 3’ direction. This means that template is read in 3’ to 5’ direction

• DNA polymerase synthesizes a new complementary strand of DNA by incorporating deoxynucleotide monophosphate sequentially in 5’ to 3’ direction

• Both strands are replicated simultaneously• Two double strands are produced in

replication. Each daughter cell gets only one strand of the parent DNA. ( semiconservative)

Steps Involved in DNA Replication in Eukaryotes

1. Identification of the origins of replication2. Unwinding ( denaturation ) of dsDNA to an

ssDNA template3. Formation of the replication fork4. Initiation of DNA synthesis and elongation

5. Formation of replication bubbles with ligation

of the newly synthesized DNA segment6. Reconstitution of chromatin structure

Classes of Proteins Involved in Replication

PROTEIN FUNCTION

DNA polymerase Deoxynucleotide polymerization

Helicases Processive unwinding of DNA

Topoisomerases Relieve torsional strain that results from helicase induce unwinding

RNA primase Initiates synthesis of RNA primer

Single-strand binding proteins Prevent premature reannealing of dsDNA

DNA ligase Seals the single strand nick between the nascent chain and Okazaki fragments on lagging strand

Origin of Replication• Specific site where replication begins ( Ori C)• With series of direct repeat DNA sequence, rich

in A-T sequence, that bind to specific protein ( Dna A )• Site at which initial separation of parental strands

occurs• 150 -250 base pairs of DNA forms complex with

multimers of DNA- binding protein (DnaA )• This leads to local denaturation and unwinding of

an adjacent A-T rich region of DNA

• In bacterial chromosome, origin of replication is single and circular, replication process is completed in 30 minutes

• In eukaryotes, origin of replication is multiple (bubbles formation ) and linear, this is to complete replication in a reasonable period of time (approx. In 9 hours)

DNA Unwinding• Unwinding of DNA, provides a short region of

ssDNAs to which SSB and helicase binds; this ssDNAs are essential for synthesis of nascent DNA strands

• Single stranded DNA binding (SSB)protein - is not an enzyme, also called helix destabilizing protein - binds only to single stranded DNA, maintains seperation, protect DNA from nucleases

• DNA Helicase - catalyzes processive unwinding of double helix of DNA - requires energy from ATP

Formation of Replication Fork

• Formed by a number of protein-protein and protein-DNA interactions

• Four components of replication that form in following sequence:

1.DNA helicase unwinds a short segment of parental duplex DNA

2.A primase initiates synthesis of an RNA molecule that is essential for priming DNA synthesis

• 3. The DNA polymerase initiates nascent daughter strand synthesis

• 4. SSBs bind to ssDNA and prevent premature reannealing of ssDNA to dsDNA

DNA Synthesis

• Template refers to structural sequence of the polymerized monomeric units of a macromolecules that provides the pattern for the synthesis of another macromolecule with a complementary or characteristic sequence

• Primer refers to a polymer molecule that contains the growing point for further addition of monomeric units

• In prokayrotes and eukaryotes, replication occurs in both strands ( bidirection )

• DNA Polymerase III –functions at replication fork, catalyzes the highest rate of chain elongation and is the most processive

• DNA Polymerase II – mostly involved in proofreading and DNA repair

• DNA Polymerase I – completes chain synthesis between Okazaki fragments on lagging strand

Comparison of Prokaryotic and Eukaryotic DNA Polymerases

E. Coli Mammalian Function

I alpha Gap filling and synthesis of lagging strand

II epsilon DNA proofreading and repair

beta DNA repair

gamma Mitochondrail DNA synthesis

III delta Processive leading strand synthesis

DNA Chain Initiation

• Initiation of synthesis requires priming by short length of RNA (10 – 200 nucleotides )

• DNA polymerase cannot initiate synthesis of new strands, requires primer

• Primer – an RNA oligonucleotide, synthesized by RNA polymerase ( PRIMASE) in a 5’ to 3’ direction, copies DNA template strands

• RNA primers – are complementary to the sequence on the strand of DNA template and base pair with that portion of DNA

• DNA polymerase initially adds deoxyribonucleotide to 3’ hydroxyl group of primer

• Process involves nucleophilic attack by the 3’ hydroxyl group of the RNA primer on the alpha phosphate of the first entering deoxynucleoside triphosphate, with splitting off of pyrophosphate

DNA Chain Elongation

• DNA polymerase elongates new DNA strand by adding deoxyribonucleotide, one at a time to the 3’ end of growing chain

• Nucleotide building blocks are 5’ deoxyribonucleotide triphosphate

• Incoming nucleotide added forms base pair with complementary nucleotide on template strand, an ester bond is formed with free 3’ hydroxyl group at end of growing chain and pyrophosphate is released

• Release of pyrophosphate and its subsequent cleavage provide the energy for the polymerization process

• Sequence is dictated by the base sequence of template strand

• All four deoxyribonucleoside triphosphates must be present for DNA elongation to occur

Direction of DNA Synthesis

• DNA polymerase reads parental nucleotide sequences in 3’ to 5’ direction and synthesizes new DNA strands in 5’ to 3’ direction

• Leading strand is strand that grows in 5’ to 3’ direction, towards replication fork, synthesized continuously

• Lagging strand is copied in 5’ to 3‘ direction away from replication fork, synthesized discontinuously with small fragment of DNA ( Okazaki fragment )

Proofreading

• Misplaced nucleotide are hydrolytically removed and replaced with correct nucleotide

• Excision is done opposite of synthesis DNA ( by3’ to 5’ exonuclease activity of polymerase )

Prevention of Supercoiling1. Type I DNA Topoisomeras - reversibly cuts a single strand of the double helix - possesses both nuclease ( strand cutting ) and ligase ( strand resealing ) activities - makes transient “ nick “ which allows DNA helix to rotate at phosphodiester bond opposite the nick - do not requires ATP2. Type II DNA Topoisomerase - make transient breaks in both DNA strands - causes a second stretch of DNA double helix to pass through the break and reseals

Termination of DNA Synthesis

• DNA polymerase III stops synthesis when it is blocked by proximity to an RNA primer

• RNA is then excised and gap is filled by DNA polymerase I

• As synthesis is being made, proofreading is also being done to new chain using 3’ to 5’ exonuclease activity

• This removal, synthesis and proofreading continues one nucleotide at a time, until RNA is totally degraded and gap is filled with DNA

• DNA ligase catalyzes formation of phosphodiester linkage between 5’ phosphate group on DNA chain synthesized by DNA polymerase III and 3’ hydroxyl group on chain made by DNA polymerase I

• This process requires ATP

Reconstitution of Chromatin Structure

• Newly replicated DNA is rapidly assembled into nucleosomes

Summary of DNA Replication

• 1. Unwinding of parental DNA to form a replication fork

• 2. RNA primer complimentary to DNA template is synthesized by RNA primase

• 3. DNA synthesis is continuous in the leading strand ( towards replication fork ) by DNA polymerase

• 4. DNA synthesis is discontinuous in lagging strand ( away from fork ) as Okazaki fragments

• 5. In both strands, synthesis is in 5’ to 3’ direction

• 6. When polymerization is complete, the RNA primer are removed, gaps filled by deoxynucleotide and strands are ligated by DNA ligase

• 7. Proof reading is done by DNA polymerae• 8. Finally organized into chromatin

DNA Synthesis Occurs During S Phase

• Replication occurs during S phase or synthetic period

• Cell regulates its DNA synthesis grossly by allowing it to occur only at specific times and mostly in cells preparing to divide by a mitotic process

• During S phase, DNA polymerase increases in quantities

• Enzymes responsible for formation of substrates for DNA synthesis ( ie, deoxyribonucleoside triphosphate) are also increased in activity and then decrease following S phase until the reappearance of signal for renewed DNA synthesis.

• During S phase, nuclear DNA is completely replicated once and only once, it marked to prevent its further replication until it again passes through mitosis

Cyclins

• Gene products that govern the transition from one phase of the cell cycle to another

• These are a family of proteins whose concentration increases and decreases throughout the cell cycle

• These are turned on appropriate time, when turned on , different cyclin dependent protein kinases ( CDKs ) phosphorylate substrate essential for progression through the cell cycle

Cyclin and Cyclin –dependent Kinases Involved in Cell Cycle Progression

Cyclin Kinase Function

D CDK4, CDK6 Progression past restriction point at G1/S boundary

E, A CDK2 Initiation of DNA synthesis in early S phase

B CDK1 Transition from G2 to M

Repair Mechanisms

• Used in persistent replication errors• Used in damages by mutagens from

environment, physical and chemical agents

Mutagens – agents that DNA damage causing mutation or cancer

Types of Damage to DNA

• I. Single base alteration A. Depurination B. Deamination of cytosine to uracil C. Deamination of adenine to hypoxanthine D. Alkylation of base E. Insertion or deletion of nucleotide F. Base-analog incorporation

• II. Two base alteration A. UV light induced thymine (pyrimidine )

dimer B. bifunctional alkylating agent cross

linkage

• III. Chain break A. ionizing radiation B. radioactive disintegration of backbone

element C. oxidative free radical formation

• IV. Cross linkage A. between bases in same or opposite

strands B. between DNA and protein molecules (eg

histone)

Mechanism of DNA Repair Mechanism Problem Solution

Mismatch repair Copying errors ( single base or two to five base unpaired loops)

Methyl directed strand cutting, exonuclease digestion and replacement

Base excision repair

Spontaneous, chemical or radiation damage to a single base

Base removal by N glycosylase, abasic sugar removal, replacement

Nucleotide excision repair

Spontaneous, chemical or radiation damage to a DNA segment

Removal of an approximately 30 nucleotide oligomer and replacement

Double strand break repair

Ionizing radiation, chemotherapy, oxidative free radicals

Synapsis, unwinding, alignment, ligation

Basic Steps DNA Repair

• 1. Recognition of distortion / damage in DNA• 2. Removal of portion / region with distortion• 3. Filling up of gap by DNA polymerase using

undamaged strand as template• 4. Sealing of nick by ligase

Mismatch Repair• Repair error made when DNA is copied • Bases are not paired correctly, or 2 to 5 extra unpaired

bases are inserted due to polymerase slip or stutter • Steps in repair: - In bacteria, a specific protein scans using adenine

methylation within GATC sequence as reference point - Template strand is methylated and newly

synthesized is not - Difference allows repair enzymes to identify strand

with errant nucleotide that requires replacement

- GATC endonuclease cuts strand with the mutation

- Faulty DNA is removed by an exonuclease - Defect is filled according to the pairing rule and

then ligatedIn mammalian or human, error can also be

distinguish and then repair mismatch but the process more complicated

Hereditary Nonpolyposis Colon Cancer ( HNPCC )-linked to faulty mismatch repair mechanism

Base Excision Repair• Repairs damage to single base ( depurination /

depyrimidination) or occurrence of not normal base

• Depurination of DNA is due to thermal lability of purine N glycosidic bond

• Bases may be deaminated to form abnormal bases, cytosine – uracil, adenine – hypoxanthine, guanine – xanthine

Steps in repair: - Abnormal base is recognized and removed by N

glycosylase

- Removal marks site of defect and allows an apurinic or apyrimidinic endonuclease to excise the abasic sugar

- Proper base is then replaced by DNA polymerase and a ligase returns DNA original base

Nucleotide Excision Repair

• Mechanism is used to repair and replace region of damaged DNA up to 30 bases in length

• Repair damaged caused by the following:1.UV light which induces formation of

cyclobutane pyrimidine-pyrimidine dimers2.Smoking which causes formation of benzo[a]

pyreo-guanine adducts

3. Other causes – ionizing radiation, cancer chemotherapeutic agents and variety of chemicals that cause base modification, strand breaks, cross linkage between bases on opposite strands or between DNA and protein

Example best studied are pyrimidine dimers formation caused by UV damage, adjacent pyrimidine residues in DNA become covalently linked ( Xeroderma pigmentosa )

• Steps in repair1.Defect is detected and unwinding of strand is

made2.Hydrolysis of 2 phosphodiester bonds in

strand defect by excision nuclease (exinuclease )

3.Fragment of DNA 27 to 20 nucleotides long is excised

• 4. strand is removed and replaced by base pairing by polymerase and then joined to existing strand by DNA ligase

Double Strand Break Repair

• Mechanism is part of physiologic process of immunoglobulin gene rearrangement

• It is also used to repair damage caused by ionizing radiation and oxidative free radical generation

• Involves 2 proteins; Ku- with latent ATP dependent helicase and DNA – PK ( DNA Protein Kinase )

• Steps in repair 1. Ku binds to free DNA ends 2. DNA-PK is recruited by KU, bind to free DNA

ends 3. There is approximation of two separate

ends 4. DNA –PK reciprocally phosphorylate Ku and

other DNA – Ku on opposite strand

• 5. DNA- PK dissociates from DNA – Ku, resulting in activation of Ku helicase

• 6. Two end unwound• 7. Unwound, approximated DNA form base

pair• 8. Extra nucleotide tails are removed by

exonuclease and gaps filled and closed by DNA ligase

Inhibitors of DNA ReplicationInhibitors of DNA replication are valuable in the treatment of various types

of diseasesUsed in antibiotics and anti cancer drugsA. Inhibitors of Nucleotide Biosynthesis - they interfere with the production of dTTP - examples are methotrexate and fluorodeoxyuridylate

B. Inhibitors that Interact with DNA Template - planar structure of drug binds noncovalently between stacked of base pairs of duplex DNA, process is called intercalation - DNA function s poor template, thus affect fidelity of replication - examples are actinomycin, acridine, ethidium

C. Nucleoside Analog Inhibitors - block further chain growth at replication fork - deoxynucleosides monophosphates are converted to triphosphates - these are incorporated into 3’ hydroxyl end of g rowing DNA chain and

because the new end lacks 3’ hydroxyl, no further addition can occur

D. Inhibitors that Bind to Replication Proteins - Aryldrazinopyrimidines are potent inhibitors of DNA polymerase III of

Gram (+) bacteria, form ternary complex with polymerase and DNA template

- Aphidicolin, a tetracyclic diterpenoid is a potent inhibitor of mammalian nuclear DNA polymerase

- Nalidixic acid and novobiocin bind to DNA gyrase to inhibit its action