Molecular GeneticsThe Central Dogma of

Biology

What is a Genetic “Factor”?

• From Mendel:– we now accepted that there was

genetic transmission of traits.• Traits are transmitted by “factors”

– Organisms carry 2 copies of each “factor”

• The question now was: what is the factor that carries the genetic information?

Requirements of Genetic Material

• Must be able to replicate, so it is reproduced in each cell of a growing organism.

• Must be able to control expression of traits– Traits are determined by the proteins that act

within us– Proteins are determined by their sequences

• Therefore, the genetic material must be able to encode the sequence of proteins

• It must be able to change in a controlled way, to allow variation, adaptation, thus survival in a changing environment.

Chromosomes – The First Clue

• First ability to visualize chromosomes in the nucleus came at the turn of the century– construction of increasingly powerful

microscopes – the discovery of dyes that selectively colored

various components of the cell • Scientists examined cellular nuclei and

observed nuclear structures, which they called chromosomes

• Observation of these structures suggested their role in genetic transmission

Chromosome Observations

• Variety of chromosome types found in the nucleus • 2 copies of each type of chromosome in most cells.• All of the cells of an organism, except gametes and

rbc’s, have the same number of chromosomes. • All organisms of the same species have the same

number of chromosomes. • The number of chromosomes in a cell doubles

immediately prior to cell division • Gametes have exactly half of the number of

chromosomes as the somatic cells of any organism.– Gametes have just one copy of each chromosome type.– Fertilization produces a diploid cell (a zygote), with the same

number of chromosomes as somatic cells of that organism.

Implications• Chromosomes behaved like Mendel’s “factors”

– Mendel's hereditary factors were either located on the chromosomes or were the chromosomes themselves.

• Proof chromosomes were hereditary factors – 1905:• The first physical trait was linked to the presence

of specific chromosomal material – conversely, the absence of that chromosome

meant the absence of the particular physical trait.

• Discovery of the sex chromosomes– "X" and "Y." – distinguished from other chromosomes and from

each other

Importance of Sex Chromosomes• Somatic cells taken from female donors always

contained 2 copies of the X chromosome • Somatic cells taken from male donors always

contained one copy of the X chromosome and one copy of the Y chromosome

• All of the other chromosomes in the nucleated cells of both male and female donors appeared identical

• Thus, gender was shown to be the direct result of a specific combination of chromosomal material– The first phenotype (physical characteristic) to be

assigned a chromosomal location – Specifically the X and Y chromosomes.

What Carries the Genetic Information?

• Chromosomes are about 40% DNA & 60% protein. – Protein is the larger component

• Protein molecules are composed of 20 different subunits

• DNA molecules are composed of only four• Therefore protein molecules could encode more

information, and a greater variety of information– protein had the possibility for much more diversity than

in DNA

• Therefore, scientists believed that the protein in chromosomes must carry the genetic information

The Discovery of DNA

• First identified in 1868 by Friedrich Miescher, a Swiss biologist, in the nuclei of pus cells obtained from discarded surgical bandages.

• He called the substance nuclein• Noted the presence of

phosphorous

The Transforming Principle• Fredrick Griffith - 1928• Discovered that different strains of the

bacterium Strepotococcus pneumonae had different effects on mice– One strain could kill an injected mouse (virulent)– Another strain had no effect (avirulent) – When the virulent strain was heat-killed and injected

into mice, there was no effect. – But when a heat-killed virulent strain was co-injected

with the avirulent strain, the mice died.

• Concluded that some factor in the heat killed bacteria was transforming the living avirulent to virulent?

• What was the transforming principle and was this the genetic material?

The Transforming Principle is DNA

• Avery, Macleod, & McCarty – 1943• Attempted to identify Griffith’s “transforming

principle”• Separated the dead virulent cells into fractions

– The protein fraction– DNA fraction

• Co-injected them with the avirulent strain.– When co-injected with protein fraction, the mice lived– with the DNA fraction, the mice died

• Result was IGNORED– Most scientists believed protein was the genetic

material. – They discounted this result and said that there must

have been some protein in the fraction that conferred virulence.

The Hershey-Chase Experiment

• Hershey & Chase – 1952• Performed the definitive experiment

that showed that DNA was the genetic material.

• Bacteriphages = viruses that infect bacteria

• Bacteriphage is composed only of protein & DNA

• Inject their genetic material into the host

The Experiment

• Prepared 2 cultures of bacteriophages• Radiolabeled sulphur in one culture

– there is sulphur in proteins, in the amino acids methionine and cysteine

– there is no sulphur in DNA

• Radiolabeled phosphorous in the second culture– there is phosphorous in the phosphate backbone of

DNA – none in any of the amino acids.

• So this one culture in which only the phage protein was labeled, and one culture in which only the phage DNA was labeled.

Experiment Summary

• Performed side by side experiments with separate phage cultures in which either the protein capsule was labeled with radioactive sulfur or the DNA core was labeled with radioactive phosphorus.

• The radioactively labeled phages were allowed to infect bacteria.

• Agitation in a blender dislodged phage particles from bacterial cells.

• Centrifugation pelleted cells, separating them from the phage particles left in the supernatant.

Results Summary

• Radioactive sulfur was found predominantly in the supernatant.

• Radioactive phosphorus was found predominantly in the cell fraction, from which a new generation of infective phage was generated.

• Thus, it was shown that the genetic material that encoded the growth of a new generation of phage was in the phosphorous-containing DNA.

Chargaff’s Rule • Once DNA was recognized as the genetic material,

scientists began investigating its mechanism and structure.

• Erwin Chargaff – 1950 – discovered the % content of the 4 nucleotides was the same in

all tissues of the same species– percentages could vary from species to species.

• He also found that in all animals (Chargaff’s rule): %G = %C

%A = %T• This suggested that the structure of the DNA was

specific and conserved in each organism. • The significance of these results was initially overlooked

Base Pairing in DNA• Not understood ‘til Watson &

Crick described double helix• Adenine & guanine are

purines– 2 organic rings

• Cytosine & guanine are pyrimidines– 1 organic ring

• Pairing a purine & a pyrimidine creates the correct 2 nm distance in the double helix

• A – T joined by 2 hydrogen bonds

• G – C joined by 3 hydrogen bonds

The Double Helix • James Watson and Francis Crick – 1953• Presented a model of the structure of

DNA. • It was already known from chemical

studies that DNA was a polymer of nucleotide (sugar, base and phosphate) units.

• X-ray crystallographic data obtained by Rosalind Franklin, combined with the previous results from Chargaff and others, were fitted together by Watson and Crick into the double helix model.

Watson and Crick shared the 1962 Nobel Prize for Physiology and Medicine with Maurice Wilkins.

Rosalind Franklin died before this date.

DNA Structure• The double helix is formed from two strands of

DNA • DNA strands run in opposite directions • They are complementary

– attached by hydrogen bonds between complimentary base pairs:

– A - T and G - C

• This complementary pairing of the bases suggests that, when DNA replicates, an exact duplicate of the parental genetic information is made. – The polymerization of a new complementary strand

takes place using each of the old strands as a template.

Messelson and Stahl• How does DNA replicate?• Matthew Meselson and Franklin Stahl - 1957• Did an experiment to determine which model

best represented DNA replication: • semiconservative replication

– the two strands unwind and each acts as a template for new strands

– each new strand is half comprised of molecules from the old strand

• conservative replication – the strands do not unwind, but somehow generate

a new double stranded DNA copy of entirely new molecules

The Experiment• The original DNA strand was labeled with

the heavy isotope of nitrogen, N-15. • This DNA was allowed to go through one

round of replication with N-14 (non-labeled)

• the mixture was centrifuged so that the heavier DNA would form a band lower in the tube

• the intermediate (one N-15 strand and one N-14 strand) and light DNA (all N-14) would appear as a band higher in the tube

The expected results for each model were:

The actual results were as expected for the semiconservative model and thus Watson and Crick's suspicion was borne out.

Enzymes & Replication• DNA replication is not a passive,

spontaneous process. • More than a dozen enzymes & other proteins

are required to unwind the double helix & to synthesize & finalize a new strand of DNA.

• The molecular mechanism of DNA replication can best be understood from the point of view of the machinery required to accomplish it.

• The unwound helix, with each strand being synthesized into a new double helix, is called the replication fork.

The Enzymes of DNA Replication

• Topoisomerase – Responsible for initiation of unwinding of

DNA.– The tension holding the helix in its coiled

and supercoiled structure is broken by nicking a single strand of DNA.

• Helicase – Accomplishes unwinding of the original

double strand, once supercoiling is eliminated by topoisomerase.

– The two strands want to bind together because of hydrogen bonding affinity for each other, so helicase requires energy (ATP) to break the strands apart.

Single-stranded Binding Proteins

• Important to maintain the stability of the replication fork.

• Line up along unpaired DNA strans, holding them apart

• Single-stranded DNA is very unstable, so these proteins bind bind to it while it remains single stranded and keep it from being degraded.

Beginning: Origins of Replication

• Replication begins at specific sites called origins of replication

• In prokaryotes, the bacterial chromosome has a specific origin

• In eukaryotes, replication begins at many sites on the large molecule– 100’s of origins

• Proteins that begin replication recognize the origin sequence

• These enzymes attach to DNA, separating the strands, and opening a replication bubble

• The end of the replication bubble is the “Y” shaped replication fork, where new strands of DNA elongate

Elongation• Elongation of the new DNA strands is catalyzed

by DNA polymerase• Nucleotides align with complimentary bases on

the template strand, and are added by the polymerase, one by one, to the growing chain – DNA polymerase proceeds along a single-stranded

DNA molecule, recruiting free nucleotides to H-bond with the complementary nucleotides on the single strand

• Forms a covalent phosphodiester bond with the previous nucleotide of the same strand – The energy stored in the triphosphate is used to

covalently bind each new nucleotide to the growing second strand

• Replication proceeds in both directions

The Replication Fork

DNA Polymerase• There are different forms of DNA

polymerase– DNA polymerase III is responsible for the

synthesis of new DNA strands • DNA polymerase is actually an

aggregate of several different protein subunits, so it is often called a holoenzyme.

• Primary job is adding nucleotides to the growing chain

• Also has proofreading activities

Proofreading & Repair

• DNA polymerase proofreads each nucleotide added against its’ template as it is added

• Removes incorrectly paired nucleotides & corrects

DNA Strands are Anti-parallel• The sugar phosphate backbones of the 2

parent strands run in opposite directions– “upside down” to each other

• DNA is polar– There is a 3’ end and a 5’ end– At the 3’ end, a OH is attached to the 3’ C of

the last deoxyribose– At the other end a phosphate group is

attached to the 5’ C of the last nucleotide• DNA polymerase adds nucleotides only

to the 3’ end of the growing chain– So new DNA strand elongates only in the

5’ 3” direction

Why 5’ 3’?• Why can DNA polymerase only add nucleotides

to the 3’ end ? • Needs a triphosphate to provide energy for the

bond between an added nucleotide & the growing DNA strand.

• This triphosphate is very unstable – can easily break into a monophosphate and an

inorganic pyrophosphate

• At the 5' end, this triphosphate can easily break– It is not be able to attach new nucleotides to the 5'

end once the phosphate has broken off

• The 3' end only has a hydroxyl group– As long as new nucleotide triphosphates are brought

by DNA polymerase, synthesis of a new strand continues, no matter how long the 3' end has remained free.

Leading & Lagging Strands• The new strand made by adding to the 3’ end

= leading strand – Parent strand is 3’ 5’– New strand is 5’ 3’

• How can DNA polymerase synthesize new copies of the 5' 3' strand, if it can only travel in one direction?

• To elongate in the other direction, the process must work away from the replication fork

• The new strand formed on the 5’ 3’ parent strand is called the lagging strand

Building the Lagging Strand

• DNA polymerase makes a second copy in an overall 3’ 5’ direction

• First, it produces short segments, called Okazaki fragments– These are built in a 5’ –3’ direction

• Okazaki fragments are joined together by ligase to produce the new 3’5’ lagging strand

Ligase• Catalyzes the formation of a

phosphodiester bond given an unattached adjacent 3‘ OH and 5‘ phosphate.

• This can join Okazaki fragments• This can also fill in the unattached

gap left when an RNA primer is removed

• DNA polymerase can organize the bond on the 5' end of the primer, but ligase is needed to make the bond on the 3' end.

Primers• DNA polymerase cannot start

synthesizing on a bare single strand. – It only adds to an existing chain

• It needs a primer with a 3'OH group on which to attach a nucleotide.

• The start of a new chain is not DNA, but a short RNA primer– Only one primer is needed for the leading

strand– One primer for each Okazaki fragment on

the lagging strand

Primase• Part of an aggregate of proteins

called the primeosome.• Attaches the small RNA primer to

the single-stranded DNA which acts as a substitute 3'OH so DNA polymerase can begin synthesis

• This RNA primer is eventually removed by RNase H – the gap is filled in by DNA polymerase

I.

Ending the Strand• DNA polymerase only adds to the 3’ end• There is no way to complete the 5’ end of the

new strand• A small gap would be left at the 5’ end of each

new strand• Repeated replication would then make the

strand shorter and shorter, eventually losing genes

• Not a problem in prokaryotes, because the DNA is circular– There are no “ends”

Telomeres• Eukaryotes have a special sequence of

repeated nucleotides at the end, called telomeres

• Multiple repititions of a short nucleitide sequence– Can be repeated hundreds, or thousands of times– In humans, TTAGGG

• Do not contain genes• Protects genes from erosion thru repeated

replication• Precvents unfinished ends from activating DNA

monitoring & repair mechanisms

Telomerase

• Catalyzes lengthening of telomeres• Includes a short RNA template with

the enzyme• Present in immortal cell lines and in

the cells that give rise to gametes• Not found in most somatic cells• May account for finite life span of

tissues

Further Proofreading & Repair

• Some errors evade initial proofreading or occur after synthesis

• DNA can be damaged by reactive chemicals, x-rays, UV, etc

• Cells continually monitor DNA for mutations & repair

• Contain 100’s of repair enzymes

Nucleotide Excision & Repair

• A segment of DNA containing damage is cut out by a nuclease (a DNA cutting enzyme)

• The gap is filled & closed by DNA polymerase and ligase

• Thymine dimers• Covalent linking of thymine bases• Causes DNA to “buckle”• One common problem corrected this way

Proteins of DNA Replication

Summary View of Replication