Molecular basis of Inheritance

• Lecture 10 is on DNA Duplication

Key Concepts

• DNA is the genetic material.
• Many proteins collaborate in DNA replication and repair.
• A chromosome comprises a DNA molecule packed with proteins.

Overview

• Hereditary information is encoded in DNA and reproduced in all cells.
• DNA directs development of biochemical, anatomical, physiological, and behavioral traits.
• The identification of inheritance molecules was a major challenge in the early 20th century.
• T. H. Morgan's group showed genes are on chromosomes and DNA and protein became candidates for genetic material.
• DNA's hereditary role was first discovered by studying bacteria and viruses.

Griffith's Experiment

• Frederick Griffith discovered the genetic role of DNA in 1928.
• Griffith experimented with pathogenic (S strain) and harmless (R strain) bacterium strains.
• Living S cells injected into a mouse cause the mouse to die.
• Living R cells injected into a mouse do not kill the mouse.
• Heat-killed S cells injected into a mouse do not kill the mouse.
• A mixture of heat-killed S cells and living R cells injected into a mouse causes the mouse to die.
• The phenomenon of transformation is now defined as genotype and phenotype change due to foreign DNA assimilation.
• The macromolecule responsible for transforming the R strain into the S strain was not identified, but it was either DNA or protein.

Bacteriophages

• Further evidence for DNA as genetic material came from bacterial virus studies.
• Bacteriophages (phages) are widely used in molecular genetics research.
• A virus is DNA or sometimes RNA enclosed in a protective, usually protein coat.

Hershey-Chase Experiment

• Alfred Hershey and Martha Chase demonstrated in 1952 that DNA is T2 phage's genetic material.
• They showed that only one of the two components of T2 enters an E. coli cell during infection.
• They concluded that the injected phage DNA provides the genetic information.
• Radioactive sulfur was used a a label on proteins infecting bacterial cells.
• A blender freed phage parts outside the cells.
• Samples are centrifuged, bacteria forms a pellet, phage parts remain suspended.
• Radioactivity was measured in both pellet and liquid.
• Radioactive DNA was found in the pellets.

Chargaff's Rules

• DNA is a nucleotide polymer with a nitrogenous base, sugar, and phosphate group.
• Erwin Chargaff reported in 1950 that DNA composition varies across species.
• This evidence made DNA a credible genetic material candidate.
• Chargaff's rules:
• The base composition of DNA varies between species.
• In any species, A and T bases are equal and G and C bases are equal.
• The basis for these rules was not understood until discovery of the double helix.

Discovery of DNA Structure

• After DNA became accepted as the genetic material, the challenge how its structure accounts for its heredity role .
• Maurice Wilkins and Rosalind Franklin used X-ray crystallography to study molecular structure.
• Rosalind Franklin pictured the DNA molecule using this technique.
• Franklin's X-ray images confirmed that DNA was helical and enabled Watson to learn the width of the helix and the nitrogenous base spacing.
• The photo pattern hinted that DNA comprises two strands, creating a double helix.

DNA Model

• In 1953, James Watson and Francis Crick developed a double-helical structure of DNA.
• Watson and Crick modeled a double helix that fit the X-ray data and DNA chemistry.
• Franklin concluded there were two outer sugar-phosphate backbones with bases paired in the molecule's interior.
• Watson's model had antiparallel backbones (subunits running in opposite directions).

Base Pairing

• Initially, Watson and Crick assumed bases paired like with like (A with A, etc.), which didn't yield uniform width.
• Pairing purines with pyrimidines resulted in uniform width matching X-ray data.
• Watson and Crick reasoned that pairings were more specific, and dictated by base structures so adenine (A) can only pair with thymine (T).
• Also guanine (G) can only pair with cytosine (C)
• The Watson-Crick model explains Chargaff's rules: in any organism, A = T and G = C.

Mechanism of DNA Replication

• Watson and Crick noted specific base pairing implied a copying mechanism for genetic material and since the two DNA strands are complementary, they are templates for building a new strand during replication.
• In DNA replication, the parent molecule divides and two new daughter strands are built based on base-pairing rules.
• Semiconservative Model: each daughter molecule has one original and one new strand.

Competing Models

• Conservative Model: parent strands rejoin.
• Dispersive Model: each strand has a mix of old and new parts.

Meselson-Stahl's Experiment

• Matthew Meselson and Franklin Stahl confirmed the semiconservative model.
• They labeled old strand nucleotides with a heavy nitrogen isotope and new strand nucleotides with a light isotope.
• The first replication created a hybrid DNA band, ruling out the conservative model.
• The second replication created light and hybrid DNA, ruling out the dispersive model.

Origin of Replication in Prokaryotes

• Copying DNA is remarkably fast and accurate and more than a dozen proteins participate in DNA replication.
• Replication begins at origins of replication, where two DNA strands separate, forming a bubble.

Origin of Replication in Eukaryotes

• A eukaryotic chromosome can have hundreds or thousands of replication origins.
• Replication proceeds in both directions from each origin, copying the entire molecule.

Replication Fork

• The replication fork at each replication bubble end is a Y-shaped area where new DNA strands elongate.
• Helicases untwist the double helix at replication forks.
• Single-strand binding proteins bind and stabilize single-stranded DNA.
• Topoisomerase corrects the overwinding strain by breaking, swiveling, and rejoining DNA strands.

Primase

• DNA polymerases catalyze new DNA elongation at replication forks.
• DNA polymerases cannot initiate polynucleotide synthesis; they can only add nucleotides to an existing 3' end.
• An RNA primase synthesizes the initial nucleotide strand.
• Primase starts an RNA chain de novo and adds RNA nucleotides one at a time using parental DNA as a template.
• The short (5-10 nucleotides) primer's 3' end starts new DNA.

DNA Polymerase

• Each nucleotide added to growing DNA is a deoxynucleoside triphosphate.
• dNTPs provide nucleotides for DNA and are like metabolic ATP, except they are deoxyribose, not ribose.
• New nucleotides added lose two phosphate groups as pyrophosphate.
• Main types of DNA polymerases: DNA polymerase III (DNA pol III), which is the builder, and DNA polymerase I (DNA pol I), which replaces the RNA primer with DNA.
• Elongation rate is about 500 nucleotides per second in bacteria and 50 per second in human cells.

Direction of Polymerization

• Because of antiparallel structure, the new DNA strand can only elongate in the 5’ to 3’ direction.
• DNA polymerases add nucleotides only to the free 3' end of a growing strand.
• DNA polymerase synthesizes continuous leading strands along one template DNA strand, moving toward the replication fork.
• To elongate the lagging strand DNA polymerase must work away from the replication fork. The lagging strand is synthesized as Okazaki fragments, joined by DNA ligase

Leading Strand Synthesis

• DNA polymerase III starts to synthesize with the RNA primer .
• The leading strand elongates continuously in the 5’ to 3’ direction as the fork progresses.

Lagging Strand Synthesis

• Primase joins RNA nucleotides into a primer.
• DNA polymerase III forming Okazaki fragment.
• DNA polymerase III detaches.
• DNA Polymerase I replaces RNA with DNA.
• DNA ligase forms bonds between new DNA and fragment 1 DNA.
• The lagging strand is complete in this region

DNA Replication Machine

• DNA replication proteins form a large complex, a "DNA replication machine".
• The DNA replication machine may be steady during replication as DNA reel in parental parental DNA and extrudes daughter DNA molecules.

Proofreading and Repair

• DNA polymerases proofread new DNA, and correct any incorrect nucleotides.
• Errors in base pairing are corrected by repair enzymes.
• A nuclease cuts and replaces damaged stretches of DNA, usually DNA damage from chemicals like cigarette smoke or radiation.
• Enzymes detect and repair damaged DNA, the thymine dimer from ultraviolet radiation distorts DNA.
• A nuclease enzyme cuts the damaged DNA strand at two points, removing the damaged section.
• Repair synthesis by a DNA polymerase fills in the missing nucleotides.
• DNA ligase seals the free end of the new DNA to the old DNA, for strand completion.
• Although proofreading and repair reduce sequence change may be permanent and inherited and these mutations drive natural selection but it is rare.

End of Replication Problem

• Eukaryotic chromosomes are linear which polymerase has with finishing problems.
• The standard machinery can't fully complete the 5' ends.
• Repeats shorten DNA molecules at ends.
• Not a problem for prokaryotes with circular chromosomes, repeated eukaryotic replication shortens daughter DNA.

Telomeres

• Chromosomal DNA molecules ends have special nucleotide sequences called telomeres.
• Telomeres don't prevent DNA shortening, but they postpone gene erosion near chromosome ends.
• Telomere shortening may be linked to aging and they may be involved in in age-related diseases

Telomerase

• Essential genes would be missing from gametes if chromosomes became shorter every cell cycle.
• Telomerase enzyme lengthens telomeres in germ cells and it overcomes telomere shortening and end-replication issues.
• Expressed by germ and early embryonic cells and but is not expressed by most somatic human cells.
• Telomerase is expressed by some controlled stem cells and is expressed by 80-90% of cancer cells.

Chromosomes

• Bacterial chromosomes consist of double-stranded and circular DNA molecules associated of protein.
• DNA is "supercoiled" and located in the nucleoid and bacteria also have independently duplicated extrachromosomal DNA fragments called plasmids.

DNA Arrangement in Prokaryotes

• Eukaryotic chromosomes are linear DNA molecules associated with protein.
• Eukaryotic DNA is mainly in the nucleus, however mitochondria/chloroplasts also have DNA independently duplicated from the nuclear genome.

Chromatin

• DNA in eukaryotic cells binds to histones that act as spools that condenses and protect the DNA molecule.
• DNA + associated proteins = chromatin.
• Nucleosome: a unit of chromatin (DNA wrapped around 8 histones).

Euchromatin and Heterochromatin

• Loosely packed chromatin in theis called euchromatin the during interphase.
• Some interphase chromatin areas like centromeres and telomeres are condensed into heterochromatin.
• It’s hard for the cell to decode genetic information because of the dense packing of heterochromatin.
• Histones can be changed chemically, which changes how chromatin is set up and affects how genes are made.

Prior to Mitosis

• All chromatin compacts extensively into visible chromosomes when cells divide.

Chromosome Painting

• Special molecular tags are used to treat human chromosomes, and each pair of homologous chromosomes looks like a different color.
• The visualization of chromosomes makes it possible to see chromosome arrangements in the interphase nucleus.
• Each chromosome has its own space during interphase.
• The two homologues of a pair are not clustered together.

Explore bacterial transformation, bacteriophage composition, Hershey-Chase experiment insights. Understand the significance of radioactive isotopes and Chargaff's rules in determining DNA's role. Identify nucleotide components and their importance.