chapter 28 dna replication, repair, and recombination

Chapter 28

DNA Replication, Repair, and Recombination

Outline• DNA Replication is Semiconservative

• General Features of DNA Replication

• DNA Polymerases

• The Mechanism of DNA Replication

• Eukaryotic DNA Replication

• Telomeres and Telomerase

• DNA Repair

• Reverse Transcriptase

Double Helix Facilitates the Accurate Transmission of Hereditary Information• Semiconservative replication:

DNA Replication

Experiment of DNA semiconservative replication

Parent DNA is labeled with 15N by growing E. Coli in 15N containing medium (15NH4Cl)

Transfer E. Coli in 14N containing medium

Look at distribution

Density-gradient equilibrium sedimentation

Meselson & Stahl Experiment

Significance of semiconservative replication

The genetic information is transferred from one generation to the next generation with high fidelity.

Replication requires separation of the two strands of double helix Hydrogen bonds between the base pairs are disrupted

Heat Acid/alkali Inside a cell is done with the help of helicases which use ATP

Dissociation of double helix is termed as melting It occurs abruptly at a certain temperature

Melting temperature (Tm):

Melting is monitored by measuring absorbance at 260 nm

DNA Replication: Melting of double helix

•The temperature at the midpoint of the transition (tm) is the melting point. It depends on

• pH • ionic strength • the size • base composition of the DNA

DNA Replication: Melting of double helix

Relationship between tm and the G+C content of a DNA

DNA Replication: Melting of double helix

DNA strands with similar sequences will form partial duplexes or hybrid with each other.

Closer evolutionary relationship between species• Similar DNA sequences • DNA hybridize

This property is used to “fish out” (clone) a similar gene from different species, if the gene sequence from a species is known.

Why human DNA hybridizes much more extensively with mouse DNA than with yeast DNA?

Annealing/hybridization

Replication: polymerization of deoxyribonucleoside triphosphates along a template

What is required?

DNA Replication

• The first DNA Polymerase (short for DNA-pol I) was discovered in 1958 by Arthur Kornberg

– received Nobel Prize in physiology or medicine in 1959

DNA Polymerase

DNA Replication

First determined DNA polymerase structure • “Klenow fragment” of E. Coli DNA

polymerase I

Structure of DNA polymerase enzymes

DNA Polymerases

• 5 structural classes– Finger and thumb domains wrap around DNA and hold it across the

enzyme’s active site

• Similar overall shape

• Similar mechanism

What DNA polymerases require for replication?

• Template– DNA polymerase is a …………………………..that synthesizes a product with a

base sequence complimentary to that of the template

• Primer– DNA polymerase requires a primer with a free 3’-hydroxyl group already base-

paired to the template.

Polymerase reaction

• Two bound metal ions participate in the reaction

• One metal ion attaches to dNTP and 3’-OH group of the primer

• Second metal ion interacts only with dNTP.

• Two metal ions bridged by carboxylate groups of two Asp residues.

Polymerase reaction

How accuracy is maintained during DNA replication?

• Binding of dNTP with correct base is favored by formation of a base pair with its partner on the template strand– H-bonds contribute to this formation

Can direct the incorporation of thymidine

•shape complimentarity

• Minor groove interactions ‒ DNA polymerases donate

2H bonds to base pairs in minor groove

‒ Hydrogen bond acceptors are present in these 2 positions for all Watson-Crick base pairs

Why shape complementarity is important?

First reason:

• Shape selectivity:

‒ Binding of dNTP to DNA polymerase induces conformational change

‒ generates a tight pocket

‒ residues lining this pocket ensure the efficiency and fidelity of DNA synthesis

Second reason:

Why shape complementarity is important?

Synthesis of RNA primer

Primase: An RNA polymerase

• Synthesizes a short stretch of RNA complimentary to one of the template

DNA strands

• Later removed by hydrolysis and replaced by DNA

How replication proceeds along the parent DNA?

• Both strands of parental DNA serve as templates.• Site of DNA synthesis called “replication fork”.

Parental DNA

Unwinding of any single DNA replication fork proceeds in one direction

Problem

The two DNA strands are of opposite polarity and DNA polymerases only synthesize DNA 5’ to 3’

Solution: DNA is made in opposite directions on each template

• Leading strand -synthesized 5’ to 3’ in the direction of the replication fork …………………..

-requires a single RNA primer

• Lagging strand -synthesized 5’ to 3’ in the opposite direction.-………………………-requires many RNA primers DNA is synthesized in short fragments called Okazaki

fragments

How replication proceeds along the parent DNA?

DNA ligase reaction:

• DNA ligase catalyzes formation of phosphodiester bond• In eukaryotes, this is and ATP-driven reaction• In bacteria, this is NAD-driven reaction

• DNA ligase seals breaks in dsDNA

How are Okazaki fragments joined?

DNA ligase mechanism

DNA ligase mechanism

Helicases separate DNA strands for replication

• Helicases utilizes energy of …………….to do so

• Typically oligomers with 6 subunits

Each subunit has P loop NTPase domain

Neighboring subunits interact closely in the ring structure

Only a single strand of DNA can fit through the center of the ring

DNA strand binds to loops on 2 adjacent subunits

How are DNA strands separated?

• Initially both domains bind ssDNA

• Upon ATP binding, ‒ Cleft between domains closes ‒ A1 domain slides along DNA

• On ATP hydrolysis‒ Cleft opens up ‒ Pulls DNA from B1 domain toward A1 ‒ dsDNA separated

Helicase Mechanism

DNA Unwinding and Supercoiling

• As helicase unwinds DNA

– the DNA in front becomes overwound

– torsionally stressed DNA double helices • fold up on themselves to form tertiary structures

Circular DNA molecules with same nucleotide sequence different linking numbers

An electron micrograph showing negatively supercoiled and relaxed DNA

Topoisomers

Linking number

It is equal to the number of times that a strand of DNA winds in the right-handed direction around the helix axis when the axis lies in a plane

The linking number for a relaxed B-DNA molecule: = the number of base pairs present/ 10.4

………….. is the number of base pairs per turn

Other Terms

• Right-handed vs Left-handed

• Important numbers Linking number (Lk)

― Must be integer― Molecules differing only in linking number are topoisomers

Twisting number (Tw): a measure of the helical winding of DNA around each other― Does not have to be integer

Writhing number (Wr): a measure of the coiling of the axis of the double helix. i.e. supercoiling― Does not have to be integer

Lk = Tw + Wr

Unstressed DNA

Linking number

Unwinding the linear duplex by two turns before joining its ends

•Two limiting conformations are possible:• The DNA can fold into a structure containing 23 turns of B helix and an

unwound loop • The double helix can fold up to cross itself

• Such crossings are called ……………..

Supercoiling

Why is supercoiling biologically important?

Supercoiled DNA has more compact shape (packaging becomes easy)

Supercoiling affects DNA’s interactions with other molecules

Dealing with supercoiling during replication

• Negative supercoils must be removed and the DNA relaxed as the double helix unwinds

• Topoisomerases introduce or eliminate supercoils• Type I Topoisomerases

– Catalyze relaxation of supercoiled DNA

• Type II Topoisomerase– Adds negative supercoils to DNA

Dealing with supercoiling during replication

They alter the linking number of DNA in a 3-step process

1. Cleave one or both strands– Type I cleaves one strand– Type II cleaves two strands

2. Passage of a segment of DNA through this break

3. Reseal DNA break

Type I Topoisomerases

• Human type I topoisomerase comprises

– Four domains around a central cavity• Diameter of 20 Å (diameter of B-DNA)• Includes a tyrosine residue (Tyr 723)

Topoisomerase I Mechanism

On binding to DNA, TopoI cleaves one strand of the DNA through a Tyr (Y) residue attacking a phosphate.

When the strand is cleaved, it rotates in a controlled manner around the other strand.

The reaction is completed by religation of the cleaved strand. This relaxes the DNA!

Topoisomerase I Mechanism

Type II Topoisomerases

A more complex mechanism cuts dsDNA

Will not be covered for Chem 361

Clinical importance of Types I and II topoisomerases

• Human topoisomerase I– Inhibited by Camptothecin, an antitumor agent

• Bacterial topoisomerase II (DNA gyrase)– Target of several antibiotics

• Novobiocin blocks binding of ATP to gyrase• Nalidixic acid and ciprofloxacin interfere with breakage and

rejoining of DNA chains– Used to treat urinary track and other infections

» Including Bacillus anthracis (anthrax)

• Coordination of enzyme activity is required for precise and rapid replication of genome.

-Requires highly processive polymerases :

Example: DNA Pol III

DNA Replication is Highly Coordinated

Structure of sliding clampIt allows the polymerase to move with DNA

DNA polymerase III synthesizes

The leading and lagging strands are synthesized in a coordinated fashion

The leading and lagging strands are synthesized in a coordinated fashion

• DNA-poly III begins synthesis of the leading strand starting from RNA primer

• Helicase unwinds DNA

• ss-binding proteins bind to the unwound strands, keeping the strands separated so that both strands can serve as templates

• Lagging synthesis more complex– DNA-poly III makes Okazaki fragments– DNA-poly I removes ………………….– DNA ligase connects fragments – DNA synthesis in eukaryotes, more complex

• DNA-poly III begins synthesis of leading strand using RNA primer

• Helicase unwinds DNA

• ss-binding proteins keep strands separated so both can be templates.

• Lagging strand synthesis more complex

Lagging strand

The leading and lagging strands are synthesized in a coordinated fashion

•The mode of synthesis of the lagging strand is more complex

•Lagging strand is synthesized in fragments

• such that 5′ → 3′ polymerization leads to overall growth in the 3′ → 5′ direction

• Yet the synthesis of the lagging strand is coordinated with the synthesis of the leading strand

The leading and lagging strands are synthesized in a coordinated fashion

How is this coordination accomplished?

•DNA polymerase III

• The holoenzyme includes two copies of the polymerase core enzyme

• The core enzymes are linked to a central structure having the subunit composition γτ2δδ′χφ

• The entire apparatus interacts with the hexameric helicase DnaB

The leading and lagging strands are synthesized in a coordinated fashion

The leading and lagging strands are synthesized in a coordinated fashion

• Okazaki fragments (RNA polymerase initiates)

• Looping the template for the lagging strand places it in position for 5’--->3’ polymerization

• DNA poly III lets go off the lagging strand after adding 1000 nucleotides

• New loop formed

• RNA primer made by primase

• Gaps filled by ……………….(it removes primers too)

In E. coli: a unique site “origin of replication” is called oriC locus

Prokaryotes: ReplicationOrigin of Replication

•The DnaA proteins bind to the five high-affinity sites in oriC

•DnaA molecules form an oligomer• a cyclic hexamer

•The DNA is wrapped around the outside of the DnaA hexamer

The binding of DnaA molecules to one another signals the start of the preparatory phase

Prokaryotes: Replication

DnaAoriC:

Preparation for replication

DnaB (hexameric helicase) + DnaC (helicase loader)

SSB

“Prepriming complex”

DnaG (primase) inserts the RNA primer

Single DNA strands are exposed in the prepriming complex

•makes single-stranded DNA accessible to other proteins

Prokaryotes: Replication

DNA pol III holoenzyme +

Prepriming complex

ATP hydrolysis within DnaA

Breakup of DnaA

(prevents additional round of replication!)

The polymerase holoenzyme assembles

Prokaryotes: Replication

Eukaryote oriC is more complex

E. Coli

• Replicates 4.6 million bp• Genetic information contained in

1 chromosome• Circular chromosome

Human diploid cell Replicates 6 billion bp 23 pairs of chromosomes must be

replicated Linear chromosome

Eukaryotes: Replication

Greatest replication problem with linear chromosomes

• Complete replication of DNA ends is difficult – polymerases act only in the 5′ → 3′ direction– the lagging strand would have an incomplete 5′ end after the removal of the

RNA primer– each round of replication would further shorten the chromosome

Telomers (from Greek: telos = end)

• Ends of chromosomes are different• Hundreds of tandem repeats of six-nucleotide sequence• One of the strands is G rich at the 3′ end, and it is slightly longer than the

other strand

• Proposed model• Single-stranded region invades duplex to form large duplex loop

Telomeres are replicated by telomerase, a specialized polymerase that carries its own RNA template

Telomerase

•contains an RNA molecule that serves as the template for the elongation of the G-rich strand

•carries the information necessary to generate the telomere sequences

DNA Damage

DNA does become damaged in the course of replication through other processes

Damage to DNA can be simple

as the misincorporation of a single base complex

chemical modification of bases chemical cross-links between the two strands of the double helix breaks in one or both of the phosphodiester backbones

Results cell death or cell transformation changes in the DNA sequence that can be inherited by future generations blockage of the DNA replication process itself

Sources of Damage: Bases can be damaged by oxidizing agents, alkylating agents, and light

• Oxidation: Reactive oxygen species – hydroxyl radical reacts with guanine to form 8-oxoguanine– 8-Oxoguanine is mutagenetic

• Deamination: potentially deleterious process– adenine can be deaminated to form hypoxanthine – mutagenic

• hypoxanthine pairs with cytosine rather than thymine

Oxidation Deamination

Sources of Damage: Bases can be damaged by oxidizing agents, alkylating agents, and light

• Alkylation: – Electrophilic centers can be attacked by nucleophiles

• N-7 of guanine and adenine form alkylated adducts

• Aflatoxin B1

– produced by molds that grow on peanuts and other foods – converted into a highly reactive epoxide by a cytochrome P450 enzyme– reacts with the N-7 atom of guanosine to form a mutagenic adduct that

frequently leads to a G–C-to-T–A transversion

Sources of Damage: Bases can be damaged by oxidizing agents, alkylating agents, and light

• Ultraviolet light: ubiquitous DNA-damaging agent– covalently links adjacent pyrimidine residues along a DNA strand– pyrimidine dimer cannot fit into a double helix

• blocks replication and gene expression

– A thymine dimer is an example of an intrastrand cross-link

-Cross-links between bases on opposite strands also can be introduced by various agents

Sources of Damage: Bases can be damaged by oxidizing agents, alkylating agents, and light

High-energy electromagnetic radiation: X-rays

• produces high concentrations of reactive species in solution

• induces several types of DNA damage • single• double-stranded breaks in DNA

DNA repair pathways:

•Mismatch repair: correction in place•Nucleotide excision repair: a stretch of DNA is removed•Base excision repair: damaged base is removed and replaced

DNA damage can be detected and repaired by a variety of systems

DNA polymerases

– able to correct many DNA mismatches produced in the course of replication

– the ε subunit of E. coli DNA polymerase III functions as a 3′-to-5′ exonuclease

Error correction by the 3′→5′ exonuclease activity of DNA polymerase

DNA polymerases • As a new strand of DNA is synthesized, it is proofread

– incorrect base slows down DNA synthesis• difficulty of threading a non-Watson–Crick base pair into the polymerase

– mismatched base is weakly bound • able to fluctuate in position

– slowdown allows time for these fluctuations to take the newly synthesized strand out of the polymerase active site and into the exonuclease active site

– the DNA is degraded, one nucleotide at a time, until it moves back into the polymerase active site and synthesis continues

Error correction by the 3′→5′ exonuclease activity of DNA polymerase

•Exonuclease activity ahead of the polymerase activity

•A mismatched base (here, a C–A mismatch) impedes translocation of DNA polymerase I to the next site.

•Sliding backward, the enzyme corrects the mistake with its 3′→5′ exonuclease activity, then resumes its polymerase activity in the 5′→3′ direction.

•Contributes to remarkable fidelity of DNA replication with an error rate of less than ……………………….

Error correction by the 3′→5′ exonuclease activity of DNA polymerase

•Mismatch-repair systems consist of at least two proteins

• MutS • for detecting the mismatch

• MutL• for recruiting an endonuclease (MutH)

• cleaves the newly synthesized DNA strand close to the lesion to facilitate repair

Mismatch-repair systems

Direct Repair

• Example: photochemical cleavage of pyrimidine dimers

• Nearly all cells contain a photoreactivating enzyme called DNA photolyase– The enzyme binds to the distorted region of DNA– Uses light energy

• the absorption of a photon by the N5,N10-methenyltetrahydrofolate coenzyme forms an excited state

– cleaves the dimer into its component bases

DNA repair enzyme:AlkA

Base-Excision Repair

Base-Excision Repair

T-dimer is repaired by three enzymes:

1. Excinuclease• detects the distortion and then cuts the

damaged DNA strand at two sites• 8 nucleotides away from the

damaged site on the 5′ side • 4 nucleotides away on the 3′

side.2. DNA polymerase I

• For repair synthesis

3. DNA ligase

Nucleotide Excision repair:

U formed by the deamination of C

• excised and replaced by C!!

Uracil Repair

Why is T instead of U in DNA?

• T or U pairs with A– Only difference: a methyl group in T

• C in DNA spontaneously deaminates forming U (100 events per day!, deamination of A and G much slower)

• Potentially mutagenic because U-A occurs rather than C-G

• Prevented by DNA glycosidase– This enzyme cuts U but does not attack T

• the -CH3 in T is a tag that distinguishes T from deaminated C

Defective repair of DNA--->Cancer

• Xeroderma pigmentosum (AR) • Extreme sensitivity to UV• Skin is dry• Keratosis • Skin cancer• Death before the age of 30!

• Defect: Excinuclease part of repair system

Mutagen Detection

• Many human cancers caused by chemicals!• Chemicals usually cause mutations

How do we identify them?• Bruce Ames developed a simple, sensitive test called “Ames” for detecting

chemical mutagens.– 0.5 microg of 2-aminoantharacene gives 11,000 colonies; only 30

colonies in its absence!

Ames test

• Thin layer of agar with salmonella bacteria cannot synthesize His

• Addition of chemical mutagen to center results in many mutations (one making bacteria synthesize His again)

• Revertants make many colonies

• What if P-450 is involved in mutagenesis?

Double stranded DNA molecules with similar sequences sometimes recombine

• DNA replication copies genetic messages as faithfully as possible

• Several biochemical processes require recombination of genetic material between two DNA molecules

• Recombination plays important role in • Making molecular diversity for Ab• Manipulating genes• Generation of “gene knockout mice”

RECOMBINATION: two DNA molecules can recombine to form new DNA molecules with segments fromboth parental molecules

DNA Replication

The genes of some viruses are made of RNA

Genes in all pro and eukaryotes made of DNA

In viruses, genes made of DNA or RNA

RNA is like DNA but:• Sugar is ribose• U instead T

– RNA can be single or double stranded

Reverse Transcription

Genetic info of RNA virus is contained in its RNA

Example: Tobacco mosaic virus which infects tobacco plantsIt consists of a single strand of RNA surrounded by a protein coatAn RNA-directed RNA polymerase copies the viral RNAInfected cells die as the virus instructs the cell to commit suicide and results in

discoloration of tobacco leaf

Example: Retrovirus Known as retroviruses because information flows BACKWORD (RNA---DNA )not

from DNA--- RNA.Includes HIV-1 as well as a number of RNA viruses that produce tumors

The genes of some viruses are made of RNAReverse Transcription

Synthesis of ssDNA complementary to ssRNA, forming a RNA-DNA hybrid.

Hydrolysis of ssRNA in the RNA-DNA hybrid by RNase activity of reverse transcriptase, leaving ssDNA.

Synthesis of the second ssDNA using the left ssDNA as the template, forming a DNA-DNA duplex.

Reverse Transcription

Viral infection of RNA virus

•Viral DNA gets incorporated into the chromosomal DNA of the host and is replicated along with the normal DNA

•Later the integrated viral genome is expressed to form viral RNA and viral proteins

Reverse Transcription