DNA and its Role in Hereditary

Basic principles of DNA replication DNA and its Role in Hereditary

DNA-The molecule • DNA—the most celebrated molecule of our time—was the subject of a 1940 to 1950 scientific race to determine the structure of this incredible molecule. • But before this, scientists needed to determine what was the composition of heritable traits ,or genes, known to lie on chromosomes.

A CHROMOSOME = both DNA and protein • Because chromosomes were made of both DNA and protein it wasn’t clear exactly what was the genetic material. • Several important experiments led to the acceptance that DNA is the material passed from one generation to the next.

Four Classic Experiments Demonstrate DNA’s Genetic Role • Griffiths • Chargaff • Avery, McLeod, and McCarty • Hershey and Chase

Four Classic Experiments Demonstrate DNA’s Genetic Role Griffiths Erwin Chargaff • Bacterial exchange of DNA • Extracts of a pathogenic strain of a virus could transform a harmless strain into a pathogenic one • Bacteria can be transformed by accepting DNA from another bacteria • Chemically determine the amount of each NT in a cell. Within a species amount of A=T & G=C , but amount of A to G varies • DNA shows variability between species but constancy of base pairs within species

Four Classic Experiments Demonstrate DNA’s Genetic Role Avery, McLeod, and McCarty Hershey and Chase • Using enzymes that can degrade DNA, RNA and protein, each biomolecule was used in an attempt to transform bacteria • DNA transforms bacteria • Protein does not • RNA does not • Radioactive labels used with in a virus, to determine location of DNA and protein in virus • DNA (with P-31) is the genetic material in while protein (with S-35) remains in the viral coat

Now we know…………… • The chemical composition of DNA was determined. • What are the chemical units making up DNA? Nuclelotides..w/Adenine, Guanine, Cytosine and Thymine

Now we know…………… • What are the chemical compounds in these units? • Deoxyribose sugar • Phosphate and • One of four basese

Now we know…………… • How are these units held together? • Phosphodiester bond made by linking the 5’ phosphate to the 3’ OH group

DNA: the Genetic Material • Once most biologists were convinced that DNA was the genetic material, a race was under way to determine how the structure of DNA could account for its role in inheritance. • What two functions must DNA perform if it is to serve as the genetic material? • 1. Replication..heredity..pass to new generation • 2. Code for protein..defines the organism

The physical structure of DNA was determined by Photographs produced by X-ray crystallography Patterns of spots indicated DNA was helical (regular repeating pattern) made of _______strands. Uniform width of 2nm--- Why would this uniform width provide information on the structure? How does this explain Chargaff’s rule?

DNA: the Genetic Material What holds together the two strands of nucleotides in the double helix? This is a very strong association-easily formed, but also easily disrupted. Hydrogen bonds What is in the middle of the helix? What is on the outside? Bases in the middle/ sugar and phosphate on outside Why are the two strands considered anti-parallel? Each strand has distinct polarity---what are these “poles”? 5’ to 3’ direction as indicated by the position of phosphate (5’) and free –OH on carbon 3 of sugar

Once Watson and Crick described the structure of DNA, it was easy to see how this structure could suggest the basic mechanism of DNA replication.

Basic Mechanism of DNA replication • Before replication the parent molecule has two complementary strands of DNA. • Each base is paired by hyrdogen bonding with its specific partner • A with T • G with C

Basic Mechanism of DNA replication • The first step in replication is the separation of the two DNA strands

Basic Mechanism of DNA replication • Each old strand now serves as a template that determines the order of nucelotides • Along “new” complementary strands nucleotides plug into specific sites along the template surface according to the base-pairing rules

Basic Mechanism of DNA replication • The nucleotides are connected to form the sugar-phosphate backbones of the new strands. • Each DNA molecule now consists of one “old “ strand and one “new” strand. • We have 2 DNA molecules identical to the one molecule with which we started

The basic principle of DNA replication is elegantly simple, but the actual process involves “complex biochemical gymnastics” Why is the process complex? When the DNA in your body replicates it must copy 6 billion bases---enough type of this size to fill 900 books 1200 pages long! And it has to copy error free. DNA replication occurs with remarkable speed and accuracy while utilizing more than a dozen enzymes and many other proteins.

Strick requirements of DNA replication • DNA replication has requirements that must be met: • 1. A DNA template. 2. A free 3' -OH group • Met by requiring a primer • Met by only growing in the 5’ tp 3’ direction • The 5’ phosphate of the incoming NT bond to the 3’ OH on a growing chain

Proteins bind to a site on the chromosome called the replication origin to initiate the process 1. Forms a replication bubble—mostly A and T at these sites a. One in a plasmid b. Many in other DNA 2. Forms a replication fork—moves in two directions

E. Coli replication fork

5. What are the enzymes and proteins involved? Helicase Single-stranded binding protein Primase DNA polymerase I and III Ligase

Problems that are met 3. Chemical mechanism of replication and anti-parallel nature of DNA create difficulties in replication – it will be semi- discontinuous. 4. For accuracy, DNA must be primed before replication will begin.

DNA exists in the nucleus as a condensed, compact structure. To prepare DNA for replication, a series of proteins aid in the unwinding and separation of the double-stranded DNA molecule. These proteins are required because DNA must be single-stranded before replication can proceed: Enzymes: • Helicases and • Single strandbinding proteins Function: • Double helix unwinds, providing single-stranded DNA templates that must be stabelized

DNA structure creates difficulties in replication – it will be semi-discontinuous • One strand can be synthesized continuously in the • 5’-> 3’ direction • LEADING STRAND • The other strand is in the WRONG direction for a 5’->3’ direction of growth. • It will be synthesized in pieces • LAGGING STRAND

The problem of the opposite strand? • Primer of RNA is synthesized on DNA (NO CHECKING REQUIRED for RNA synthesis) • DNA is synthesized in the 5-> 3direction away from primer The opposite strand is backwards….

DNA structure creates difficulties in replication – it will be semi-discontinuous Synthesis of Leading Strand • Priming (primase) adds RNA • Elongation (DNA Polymerase) • Replacement of RNA by DNA (DNA Polymerase) Synthesis of Lagging strand • Priming for Okazaki framgents (primase) • Many RNA primers • Elongation of fragment (DNA polymerase) • Replacement of RNA by DNA (DNA Polymerase) • Joining of fragment (ligase)

Main enzyme is DNA polymerase DNA Polymerase 3 enzyme that performs the 5'-3' polymerase function. There are three activities associated with DNA polymerase 1 • DNA polymerase III will synthesize a continuous or discontinuous strand of DNA on both leading & lagging strand. • DNA polymerase III has a high processivity & synthesizes DNA very quickly 1. 5' to 3' exonuclease (removes primer & repair activity). 2. 3' to 5' exonuclease (proof-reading activity). • 3. 5' to 3' elongation (polymerase activity)

Events at one replication fork (usually occurs simultaneously at two forks) • Helicases unwind the parental double helix • Single-stranded binding proteins stabilize the unwound parental DNA • The leading strand is initiated by an RNA primer, as is the first Okazaki fragment • The leading strand is synthesized continuously in the 5’ 3’ directions by DNA polymerase III • The lagging strand is synthesized discontinuously. Primase synthesizes a short RNA primer, which is extended by DNA polymerase III to form an Okazaki fragment. • With 5’  3’ exonuclease and polymerase activity, the RNA primer is replaced with DNA by DNA polymerase I • DNA ligase joins the Okazaki fragment to the growing strand.

DNA Replication • http://www.youtube.com/watch?v=dIZpb93NYlw • http://www.youtube.com/watch?v=-mtLXpgjHL0&feature=related • http://www.youtube.com/watch?v=nIwu5MevZyg&feature=related

Another problem with replication • The ENDS of DNA— • The telomeres! • An RNA primer is not synthesized on the end of the lagging strand

Another problem with replication • A chromosome has stretches of noncoding regions • At the centromere and • the telomeres (at the ends of the chromosome). • Telomeres are crucial to the life of the cell. • They keep the ends of the various chromosomes in the cell from accidentally becoming attached to each other.

Another problem with replication • The telomeres of humans consist of as many as 2000 repeats of the sequence 5' TTAGGG 3‘ • 5'...TTAGGG TTAGGG TTAGGG TTAGGG TTAGGG TTAGGG..3' • 3'...AATCCC AATCCC AATCCC AATCCC AATCCC AATCCC 5’

It is estimated that human telomeres lose about 100 base pairs from their telomeric DNA at each mitosis. This represents about 16 TTAGGG repeats. At this rate, after 125 mitotic divisions, the telomeres would be completely gone. Is this why normal somatic cells are limited in the number of mitotic divisions before they die out?

Telomeres and Cellular Aging • Telomeres are important • Steadily shrinking with each mitosis…. imposes a finite life span on cells. • Normal (non-cancerous) cells do not grow indefinitely when placed in culture.

Problems: • 1. Lagging strand w/ improper orientation • 2. Telomers

Using DNA Replication • 1. PCR • 2. Sequencing • 3. Drug therapy

Using DNA Replication • PCR-----That’s a another SLIDE show • The polymerase chain reaction (PCR) • a technique to amplify a specific piece of DNA across several orders of magnitude • The polymerase chain reaction (PCR) enables researchers to produce millions of copies of a specific DNA sequence in approximately two hours

Using DNA Replication- Drug therapy • Target the 3’OH group • AZT …azidothymidine, also called zidovudine • drug used to delay development of AIDS in patients infected with HIV • AZT belongs to a group of drugs known as nucleoside analogs

Using DNA Replication:Antimetabolites A class of drugs that interfere with DNA and RNA growth by substituting for the normal building blocks of RNA and DNA. Used to treat leukemias, tumors of the breast, ovary, and the intestinal tract, as well as other cancers. • Examples • 5-fluorouracil (5-FU) • 6-mercaptopurine (6-MP),,

Using DNA replication-Sequencing • Nestled sizes of DNA—each one NT shorter than the other • Target the 3’ OH group • Dideoxynucleotides • Labeled NT’s

Newer techniques use fluorescent dyes that read DNA fragments as they run off of a gel.

Using DNA Replication as a TOOL • REPLICATION AND SEQUENCING ANIMATION

DNA and its Role in Hereditary