Mrs. OFELIA SOLANO SALUDAR

Department of Natural SciencesUniversity of St. La Salle

Bacolod City

Tommy was a full-term baby but weighed only 4.5 pounds (2 kg) at birth. At about 9 months of age, an unusual and persistent rash appeared on his face, and he frequently caught colds and infections. The illnesses caused no serious problems; so his parents were not concerned.

Throughout childhood, Tommy remained small; by age 18, he was only 4 feet 6 inches (137 cm) in height. Tommy’s first major health problem arose shortly after he turned 22—he was diagnosed with intestinal cancer. The tumor was surgically removed but additional, unrelated tumors appeared spontaneously over the next 10 years.

Their appearance startled Tommy’s doctors; the chance of multiple, independent cancers arising in the same person is generally remote.

The propensity of Tommy’s cells to become cancerous hinted at a high mutation rate in his genes. Indeed, when pathologists studied Tommy’s chromosomes, they observed a wide range of abnormalities. Tommy had inherited BLOOM SYNDROME.

Bloom syndrome is a rare autosomal recessive condition characterized by short stature, a facial rash induced by sun exposure, a small narrow head, and a predisposition to cancers of all types.

The disorder is extremely rare; only several hundred cases have been reported worldwide. Cells from persons with Bloom syndrome exhibit excessive mutations in all genes, and numerous gaps and breaks occur in chromosomes that lead to extensive genetic exchange in cell division. Rates of DNA synthesis are retarded.

The characteristics of Bloom syndrome suggest that its underlying cause is a defect in DNA replication. In 1995, researchers at the New York Blood Center traced Bloom syndrome to a gene on chromosome 15 that encodes an enzyme called DNA helicase. A variety of helicase enzymes are responsible for unwinding double-stranded DNA during replication and repair.

The cells of a person with Bloom syndrome carry two mutated copies of the gene and possess little or no activity for a particular helicase. Normal DNA replication is disrupted, leading to chromosome breaks and numerous mutations. The genetic damage resulting from faulty DNA replication leads to tumors.

It is not yet clear whether the basic defect in DNA synthesis is associated with replication or DNA repair or both.

To understand Tommy’s case, we need to answer the following questions:

What models of DNA replication exist among life forms?

Where is the origin of replication in the DNA strand?

What is the direction of replication at this site? How does the chain grow in length? How does the chain terminate? What is the enzymology behind DNA

replication? Are there other protein factors that must be

present? What is the role of DNA replication in the

expression of disease?

These models may differ with respect to the initiation and progression of replication, but

all produce new DNA molecules by semi-conservative replication.

MODELS OF DNA REPLICATION

THETA REPLICATION: E. colihttp://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120073/micro03.swf::Bidirectional%20Replication%20of%20DNA

ROLLING CIRCLE: Viruses and F factor of E. Coli

LINEAR REPLICATION

FUNDAMENTAL RULES OF DNA REPLICATION 1. Replication is semi-conservative

Meselson and Stahl convincingly demonstrated that each E. coli DNA strand serves as a template

for the synthesis of a new DNA molecule.

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2. Replication begins at an origin- the replication

fork

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3. DNA replication is bi-directional, and proceeds in a 5’-3’ direction

DNA synthesis takes place simultaneously but in opposite directions on the 2 template

strands.

Leading strand

lagging strand

Replication fork

Replication fork

4. DNA Replication is Semi-discontinuous

The polarity of DNA

synthesis creates an asymmetry

between the leading strand and the lagging strand at

the replication

fork

Although the process of replication includes many components, they can be combined

into three major groups:1. a template consisting of single-stranded

DNA,2. raw materials (substrates) to be

assembled into a new nucleotide strand, and

3. enzymes and other proteins that “read” the template and assemble the substrates into a DNA molecule.

Requirements of Replication

New DNA is synthesized from deoxyribonucleotide triphosphates (dNTPs). Since the 5’ end does not get added to and the 3’ end repeatedly does, the DNA strand is said to grow in a 5’- 3’ manner.

Components required for replication

DNA

Polymerase

DNA Polymerase III

DNA Polymerases in E. coli

Topoisomerase

Protein complexes of the replication

fork

DNA helicase unwinds

the DNA duplexahead of DNA polymerase

creating single stranded DNA

that can be usedas a template

ssDNA binding proteins bind to the sugar phosphate backbone leaving the bases exposed for DNA polymerase.

The binding of SSB to newly formed ssDNA prevents reassociation of the single strands and “iron out” the

unwound DNA.

Since DNA polymerase requires a template and

a free 3’ OH group to add nucleotides on to,

RNA primers are required to initiate DNA

polymerization. Primase, an enzyme

which is part of a large complex of proteins

called the primosome, synthesizes a small stretch of RNA (the

primer) of 3-10 nucleotide in length, which will act as a

starting site for the DNA polymerase.

DNA polymerase falls off the DNA

easily. A “sliding clamp” is required to

keep DNA polymerase on and allow duplication of

longstretches of DNA

A “clamp loader:” complex is required to get the

clamp onto the DNA

Ahead of the replication

fork the DNA becomes

supercoiled

The supercoiling needs to be relieved or tension

would build up (like coiling as spring) and

block fork progression.

Supercoiling is relieved by the action of Topoisomerases.

1. Type I topoisomerases: Make nicks in one DNA strands Can relieve supercoiling

2. Type II topoisomersases or DNA gyrase Make nicks in both DNA strands (double

strand break) Can relieve supercoiling and untangle

linked DNA helices Both types of enzyme form covalent

intermediates with the DNA

Type I Topoisomerase

Type II Topoisomerase

Topoisomerases as drug targets

1. Dividing cells require greater topoisomerase activity due to increased DNA synthesis

2. Topoisomerase inhibitors which act by stablilizing the DNA-topoisomerase complex are used as chemotherapeutic agents: camptothecin -Topo I inhibitor

doxorubicin -- Topo II inhibitor Some antibiotics are inhibitors of the

bacterial-specific topoisomerase DNA gyrase: e.g. ciprofloxacin

DNA

LIGASE

Replication is extremely accurate, with less than one error per billion nucleotides. This accuracy results from the processes of nucleotide selection, proofreading, and

mismatch repair.

DNA mismatch repair corrects errors made during DNAreplication.(A) If uncorrected, the mismatch will lead to a

permanent mutation in one of the two DNA molecules produced by the next round of DNA replication. (B) If the mismatch is “repaired” using the newly synthesized DNA

strand as the template, both DNA molecules produced by the next round of DNA replication will contain a mutation. (C) If the mismatch is corrected using the original template (old)

strand as the template, the possibility of a mutation is eliminated. The scheme shown in (C) is used by cells to repair

mismatches.

Chemical modifications of nucleotides, if left unrepaired, produce mutations.

(A) Deamination of cytosine, if uncorrected, results in the substitution of one base for another when the DNA is replicated. Deamination of cytosine produces uracil. Uracil differs from cytosine in its base-pairing properties and preferentially base-pairs with

adenine. The DNA replication machinery therefore inserts an adenine when it encounters a U on the template strand. (B) Depurination, if uncorrected, can lead to the loss of a nucleotide pair. When the replication machinery encounters a missing

purine on the template strand, it can skip to the next complete nucleotide, thus producing a nucleotide deletion in the newly synthesized strand. In other cases, the replication machinery places an incorrect nucleotide across from the missing base,

again resulting in a mutation.

SUMMARY OF STEPS OF DNA REPLICATION1. Helicase enzyme unwinds DNA.  This reaction needs ATP.  At each replicating

fork, the exposed single-stranded DNA is protected by single-strand binding proteins (ssb).  Primase enzyme binds, preparing to make RNA primers.

2. Primase enzyme makes RNA primer molecules.  Each primer hybridizes (base pairs) with DNA, at the origin of replication.  The 3' OH end will attach new deoxy nucleotides (dNTPs).  The primers will each start a leading strand,

3. DNA polymerase III attaches new dNTPs to the 3' OH end of the growing chain of the leading strand, which elongates toward the replicating fork, 5' to 3'.  (For each origin, there are TWO leading strands)  For each NTP, a pyrophosphate (PP) is released, providing the necessary energy.

4. More primers hybridize to the opposite strand of DNA.  Pol III starts elongating 5' to 3' but it keeps running into the back of an RNA primer.  This is the lagging strand.  There are TWO lagging strands.

5. DNA polymerase I (Pol I) starts at “nicks” in the growing strands.  It edits the strand by removing bases ahead of it (5' end), including RNA and mismatched bases, while elongating the strand "behind" 5' to 3'.  It replaces all RNA nucleotides with dNTPs.

6. Ligase seals the phosphate bonds at all “nicks” in the DNA. 7. Editing endonucleases excise mismatched nucleotides, replacing with the

proper match.  How do they know which is old DNA vs. new DNA?  The old DNA contains methyl groups on some of its cytosine bases.

8. Gyrase restores negative superturns in DNA.  ATP is needed. 9. Methylases add methyl groups to the new DNA, at the same positions as the

original strands.  Now the two daughter helices are indistinguishable from each other, and from the original helix.

Time for DNA replication is limited in the S phase of eukaryotes (6-8 hrs in mammals. Such RFs move

only about 1/10th of the prokaryotic forks, and chromosomes can be in excess of 108 bp.

Completion of replication at the allotted time requires multiple RFs called replicons. The Origin

Recognition Complex (ORC) is a complex of 6 ATPases which is the functional equivalent of DnaA.

THE EUKARYOTIC REPLICON

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EUKARYOTIC DNA POLYMERASES

How does a linear

chromosome close

replication at its two

ends?

As DNA synthesis requires a RNA primer that will eventually be

digested away, standard DNA

replication would result in linear

chromosomes that would shrink with

every round of replication. This is

resolved in bacteria by the circular

genome which does not have an end. In

linear chromosomes, the telomere solves the DNA end replication

problem.

Telomeres have highly repeated DNA sequences 5'-TTAGGG-3'.

Human chromosomes have between 100 and 1500 copies of this sequence.

Telomerase, a special DNA polymerase, can add additional copies of the 5'-TTAGGG-3' to the end of a chromosome.

The telomerase enzyme is actually a complex containing protein and RNA (a "ribozyme").

The RNA portion has a 5'-CCCTAA-3' region that acts as a template for adding the DNA repeat to the chromosome ends.

The telomerase enzyme is found mostly in the germ cells of multicellular organisms.

In somatic cells, the absence of telomerase results in shorter chromosomal ends with each division and may be the limiting factor in an organism's life span.

TELOMERASE AND DISEASE

Errors of DNA Replication and Disease

Origins or replication are strictly controlled so that they “fire” only once per cell cycle

Errors lead to over-replication of specific chromosomal regions = gene amplification This is commonly seen in cancer cells

and can be an important prognostic indicator.

It can also contribute to acquired drug resistance, e.g. Methotrexate induces amplification of the dihydrofolate reductase locus.

The rate of misincorporation of bases by DNA polymerase is extremely low, however repeated sequences can cause problems.

In particular, trinucleotide repeats cause difficulties which can lead to expansion of these sequences.

Depending where the repeat is located, expansion of the sequence can have severe effects on the expression of a gene or the function of a protein.

Looping out of repeats before replication.

Several inherited diseases are associated with expansion of trinucleotide repeat sequences.

Very different disorders, but they share the characteristic of becoming more severe in succeeding generations due to progressive

expansion of the repeats

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