Download Chapter 16 - Molecular Basis of Inheritance DNA as the Genetic

Chapter 16 - Molecular Basis of Inheritance
DNA as the Genetic Material
Genes are located on chromosomes
two constituents of chromosomes
proteins - more heterogeneous, thought to be genetic material (until 1940s)
DNA - originally thought to be too simple structure to be genetic material
DNA associated with proteins is chromatin
However, this was not consistent with experiments with microorganisms, like bacteria and
viruses
Griffith’s Experiment
Figure 16.2 Transformation of bacteria
non-pathogenic R strain changed to pathogenic S strain by something in heat-killed S strain
sample
But identity of transforming agent not identified
Transformation = a change in:
genotype (genetic makeup) and
phenotype (appearance)
due to the assimilation of a foreign substance (now known to be DNA) by a cell.
Key Experiment #2
-For 14 years scientists tried to identify the transforming substance
-Avery, McCarty, & MacLeod (1944)
-Identified DNA as the transforming agent
Avery et al.
purified various classes of molecules from heat-killed S strain bacteria
added to R strain (non-pathogenic)
tested for conversion to pathogenicity
• showed DNA to be transforming agent
• much resistance to idea
-genes of bacteria not thought to be similar in composition and function to those of more
complex organisms
-also couldn’t imagine how DNA could contain genetic info
Key Experiment #3
Alfred Hershey and Martha Chase (1952)
DNA was genetic material of bacteriophage T2
phage T2 attacks Escherichia coli, a common intestinal bacteria of mammals
converts E. coli cells into phage-producing factories which release phage when cell ruptures
T2 mostly composed of protein & DNA
Labeled phage T2 proteins or DNA
protein - radioactive sulfur
DNA - radioactive phosphorus
Infected E. coli cultures to determine where the radioactivity is
Fig 16.4 Hershey-Chase Experiment
Additional Evidence for DNA as Genetic Material
Mitosis
DNA content doubles prior to onset
DNA as chromosomes is divided equally between daughter cells
Diploid cells have twice as much DNA as haploid cells
Chargaff’s Rule
Erwin Chargaff (1947)
developed a series of rules based on a survey of base composition of DNA in organisms
Base composition of DNA varies from one species to another
Amounts of bases are not equal, but are present in a characteristic ratio
Chargaff’s Rule
%A = %T and %G = %C
% Purine = % Pyrimidine
(A + G = C + T)
developed before double helix structure known
In human DNA:
A & T = ~30% for each
C & G = ~ 20% for each
DNA Structure
composition of DNA was known
race to determine structure heated up in 1950s
Linus Pauling in California
Maurice Wilkins & Rosalind Franklin in London
James Watson and Francis Crick in Cambridge won the race even though they were
relatively unknown at the time
Fig 16.5 The Structure of a DNA Strand
Polymer of nucleotides
• nitrogenous base
• 2’ deoxyribose sugar
• phosphate
• Phosphate group of one nucleotide is attached to sugar of next nucleotide in line.
• Result is a “backbone” of alternating phosphates and sugars, from which bases project
Fig 16.6 X Ray Crystallography of DNA
Maurice Wilkins and Rosalind Franklin used X-ray crystallography to study structure of DNA
James Watson learned from their research that DNA was helical in shape and he deduced
the width of the helix and the spacing of bases.
Fig 16.7a The Double Helix
The key breakthrough came when Watson put the sugar-phosphate chain on the outside and the
nitrogen bases on the inside of the double helix.
The sugar-phosphate chains of each strand are like the side ropes of a rope ladder.
Pairs of nitrogen bases, one from each strand, form rungs.
The ladder forms a twist every ten bases
Fig 16.7b The Double Helix
Complementary Base Pairs
A = T G = C Purines pair with pyrimidines
Fig 16.7c The Double Helix
Base pairing in DNA (see p. 310)
paired in specific combinations
A with T and G with C (explains Chargaff’s rules)
Pairing “like” nucleotides did not fit the uniform diameter indicated by the X-ray data.
A purine-purine pair would be too wide and a pyrimidine-pyrimidine pairing would be too
narrow.
Only a pyrimidine-purine pairing would produce the 2-nm diameter indicated by the X-ray data
Fig 16.8 Base pairing in DNA
DNA Structure
In April 1953, Watson and Crick published a succinct, one-page paper in Nature reporting their
double helix model of DNA
“We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.). This
structure has novel features which are of considerable biological interest……
From Nature Vol. 171, No. 4356, pp. 737-738
Base Pairing and Sequence
Base-pairing rules:
dictate the combinations of nitrogenous bases that form “rungs” of DNA
However, base-pairing rules do not restrict the sequence of nucleotides along each DNA strand.
The linear sequence of the four bases can be varied in countless ways.
Each gene has a unique order of nitrogen bases.
Genetic information is stored in sequence of nitrogen bases
DNA Replication
The structure of DNA provided insight to Watson and Crick for how DNA replicates
Complementarity of strands
Strands form templates
order of bases on one strand can be used to add in complementary bases on other strand, and
therefore duplicate the pairs of bases exactly.
In their paper, they stated:
“It has not escaped our notice that the specific pairing we have postulated immediately
suggests a possible copying mechanism for the genetic material…..
From Nature Vol. 171, No. 4356, pp. 737-738
Fig 16.9 Model for DNA Replication
Fig 16.10 Three models of DNA replication
Experiment which supports the Semiconservative Model
Matthew Meselson and Franklin Stahl
experiments supported semiconservative model
Labeled nucleotides of old strands with a heavy isotope of nitrogen (15N) while any new
nucleotides would be indicated by a lighter isotope (14N).
Replicated strands could be separated by density in a centrifuge.
Meselson-Stahl Experiment supports the Semiconservative Model
Each model: the semi-conservative model, the conservative model, and the dispersive model,
made specific predictions on the density of replicated DNA strands.
Fig 16.11 Meselson-Stahl experiment
Conclusion: DNA replication follows semiconservative model
Conservative and Dispersive models were disproven in their experiment.
DNA Replication
More than a dozen enzymes and other proteins participate
E. coli can replicate 4.5 x 106 base pairs bp) in less than an hour
human cells can replicate 6 x 109 bp in only a few hours
DNA replication is very accurate
less than 1 error per billion nucleotides!!
DNA Replication Start Sites
Where does DNA replication start?
special sites termed origins of replication
single site in bacterial chromosome
multiple sites in eukaryotic chromosome
Enzymes (helicases) separate two strands
forms a replication “bubble”
other proteins (single strand binding proteins - ssb) bind to keep strands separated
Fig 16.12 Origins of Replication
Enzymes of DNA Replication
DNA polymerases
synthesize DNA by adding a nucleotide that is complementary to the base in the template
strand
Rate of synthesis
Bacteria - 500 nucleotides / sec
Human cells - 50 nucleotides / sec
Fig 16.11 Incorporation of a nucleotide
Fig 16.13 The two strands of DNA are antiparallel
On each strand:
5’end = PO4
3’end = OH
DNA Polymerases
Add nucleotides to free 3’ end of a DNA strand (can’t start synthesis without a 3’OH)
New strand elongates in a 5’  3’ direction
What happens during replication since the strands are antiparallel?
Fig 16.14 DNA Synthesis
leading strand is synthesized continuously
lagging strand is synthesized discontinuously in short segments called Okazaki fragments
DNA ligase joins the fragments
Initiating DNA Synthesis
After separation of the DNA strands
DNA Polymerase cannot initiate DNA syn.
Needs a 3’ OH to add nucleotide to.
synthesizing a new chain requires a primer, a short segment of RNA
Primase (an RNA Polymerase) adds about 10 nucleotides complementary to template
Fig 16.15 Priming DNA Synthesis
Note: RNA primer is removed from DNA by another DNA Polymerase
Table 16.1 Bacterial DNA Replication Proteins
Helicase - Unwinds parental double helix at replication forks
ssb proteins- bind to and stabilize ssDNA
Topoisomerase - Corrects “overwinding” ahead of replication forks; breaks, swivels, and rejoins
DNA strands
Primase - synthesizes single primer for leading strand; synthesizes RNA primer for each lagging
strand
DNA pol III - continuous synthesis of leading strand; discontinuous synthesis of lagging strand
DNA pol I - removes primer (RNA) from DNA strand and replaces it with DNA
DNA Ligase - joins 3’ end of fragment with 5’ end of adjacent fragment
Fig 16.16 Summary of DNA Replic.
DNA Replication
Single large complex (replisome)
leading and lagging strand polymerases
other proteins (helicase, primase, etc.)
Complex is stationary
DNA molecule is “reeled in” during replication
Lagging strand is looped through complex
must dissociate at end of Okazaki fragment synthesis and reassociate for synthesis of next
fragment
DNA “proofreading”
Mispairing of bases during synthesis occurs at a rate of 1/100,000 bp
DNA Polymerase “proofreads” each nucleotide added to new DNA strand
If incorrect, DNA Polymerase removes and resumes synthesis
Other Types of Repair of DNA
Bases may be damaged by chemical and/or physical agents
UV light, reactive chemicals, radiation, etc.
Some mismatched bases may be missed by proofreading activity of DNA pol
Must be corrected to ensure high fidelity of DNA sequence
DNA “Mismatch” Repair
In mismatch repair, special enzymes fix incorrectly paired nucleotides.
A hereditary defect in one of these enzymes is associated with a form of colon cancer.
hereditary nonpolyposis colorectal cancer (also known as HNPCC or Lynch Syndrome)
Nucleotide Excision Repair
Most common form of repair of DNA damage
segment of DNA strand containing the damage is excised or cut out
leaves a gap to be filled by a DNA pol
Fig 16.17 Nucleotide Excision Repair
In nucleotide excision repair, a nuclease cuts out a segment of a damaged strand.
The gap is filled in by DNA polymerase and ligase.
Xeroderma Pigmentosum
Individuals with this genetic disease have defective repair enzymes
can’t remove thymine dimers caused by UV light
very sensitive to sunlight and often get skin cancers
Summary of DNA Repair
Each cell continually monitors and repairs its genetic material, with over 130 repair enzymes
identified in humans.
The final error rate is only one per billion nucleotides, so, about 6 mutations per cell division!
Replication of Chromosome Ends
Limitations in the DNA polymerase
problems for the linear DNA of eukaryotic chromosomes.
no way to complete the 5’ ends of daughter DNA strands.
Repeated rounds of replication produce shorter and shorter DNA molecules.
Fig 16.18 The End-Replication Problem
5’ end of new strand can’t be replicated
• When it serves as a template, the resulting duplex is shorter
Telomeres at Chromosome Ends
Eukaryotic chromosome ends have special nucleotide sequences called telomeres
TTAGGG sequence repeated 100-1000 times
Protect genes near ends by preventing shortening of chromosomes during multiple rounds of
replication
Telomerase Activity
Telomerase contains a short RNA that functions as a template for extending the DNA strand
Primase and DNA Pol can now synthesize additional sequence
Telomeres
Not active in most cells of multicellular organisms.
Therefore, the DNA of dividing somatic cells and cultured cells does tend to become
shorter.
Thus, telomere length may be a limiting factor in the life span of certain tissues and the
organism.
Telomerase
Is active in germ-line cells, ensuring that zygotes have long telomeres.
Active telomerase is also found in cancerous somatic cells.
This overcomes the progressive shortening that would eventually lead to self-destruction
of the cancer.
2009 Nobel Prize in Medicine
Drs. Elizabeth Blackburn, Carol Greider, and Jack W. Szostak
“For the discovery of how chromosomes are protected by telomeres and the enzyme telomerase”