DNA Structure and replication

Genome

the complete set of genetic instructions found within a cell

Histone

proteins found in eukaryotic cell nuclei that act as microscopic spools. DNA wraps tightly around them to form structural units called nucleosomes, allowing massive lengths of DNA to fit into a tiny cell nucleus and protecting it from damage

Pyrimidine

Single-ringed structures that always pair with purines

Purine

Double-ringed structures that always pair with pyrimidines 

Chromosome

a thread-like structure made of tightly coiled DNA and proteins, located inside the nucleus of cells

Telomere

a repetitive section of DNA at each end of a chromosome

Chromatin

the highly organized, dynamic complex of DNA, RNA, and proteins (primarily histones) that makes up chromosomes in the cells of humans and other eukaryotic organisms

Nucleotide

Nucleic acids are polymers made up of many units/monomers

Made up of a phosphate group, sugar, and ntirogenous base(A,C,T,G,C)

Replication bubble

Once the enzyme has opened the molecule an unwound oval-shaped area known as the replication bubble forms.

RNA primer

short sequences (10-60 base pairs) of RNA, attached by the enzyme primase

• are used to build the lagging strand

DNA polymerase I

Removes the RNA primers on both the leading and lagging strands and replaces them with the complementary deoxyribonucleotides (DNA nucleotides)

Telomerase

Some cells have the ability to reverse telomere shortening through the action of telomerase

How does telomerase work

• extends the telomeres of chromosomes so that after each cell division they are “restored”

• It contains its own internal RNA template

• When it binds to the telomere, it extends the 3’ single-stranded overhang on the parental DNA strand with more of the telomere sequence (TTAGGG)

Nucleosome

Groups of 8 histones with DNA double helix wrapped around it

• Nucleosomes are connected by linker DNA

Anti-parallel

• At the end of a DNA molecule, the 5’ of one strand of DNA lies across from the 3’ end of the complementary strand

• The 5’ and 3’ come from the numbering of the carbon atoms on the deoxyribose sugar. 

  • The phosphate group is on the 5’ carbon, and the   -OH group is on the 3’ carbon. 

• By convention, the sequence of a DNA strand is always written/read in the 5’ to 3’ direction 

RNA primase

SSB proteins

bind to the open DNA strands to prevent them from annealing back together 

→ now hydrogen bonds can’t reform between adjacent bases, this keeps the strands apart

Gyrase

The enzyme topoisomerase II (also called DNA gyrase) relieves the tension brought about by the unwinding of the DNA strands.

Exonuclease

enzymes that break the phosphodiester bond between the nucleotides and release them, they will then be replaced with the correct nucleotides

Semiconservative

Semiconservative replication = Each parent DNA strand serves as a template for the synthesis of a new DNA molecule.  This new DNA consists of one new and one parent strand of DNA.

Replication fork

The replication fork is the junction between the unwound part and the open part. 

Phosphodiester bond

strong covalent linkages that form the fundamental backbone of DNA and RNA

Okazaki fragments

• Because fragments extend away from the replication fork, as they lengthen, new primers must be added as replication proceeds

→ Multiple RNA primers are needed for the lagging strand

• Newly synthesized DNA fragments are called Okazaki fragments

DNA polymerase III

 specifically responsible for synthesizing the new DNA strands during replication

How does DNA polymerase III work

• DNA polymerase III cleaves off two phosphates from the nucleoside triphosphate, releasing energy, and the resulting nucleotide is added to the DNA strand, creating the alternating sugar-phosphate backbone.

• can only add a nucleotide to the free 3’ hydroxyl end of an elongating DNA strand

• can attach and start adding the DNA nucleotides towards the replication fork

Origin of replication

a specific nucleotide sequence on the DNA where replication begins

Topoisomerase

Supercoiling is controlled by two enzymes : topoisomerase I and II

Leading strand

refers to the new DNA strand that is built continuously in one piece towards the replication fork (follows DNA helicase)

Lagging Strand

refers to the new DNA strand that is built in short fragments away from the replication fork

Helicase

• enzyme unzips the double helix by breaking the hydrogen bonds between the bases. 

DNA Ligase

Joins the short Okazaki fragments on the lagging strand together by creating a phosphodiester bond in the DNA backbone between adjacent sugar and phosphate

Miescher discovery and method

• He isolated nuclei_____ of white blood cells from hospital bandages (pus cells)

• Noticed that the nuclei of the cells contained large amounts of something that wasn’t protein an it contained large amounts of phosphorus

Called this substance “nuclein”, since it was mostly found in the nucleus

Hammerling discovery and method

Through experiments using the algae Acetabularia he showed that the hereditary information is found in the nucleus

Griffith discovery and method

• 1928: bacterial pneumonia (caused by Streptococcus pneumoniae bacteria) was a huge problem → Frederick Griffith: studied the pathology of the disease

• His experiments showed the phenomenon of transformation

  • Transformation = when a bacterium takes up a piece of DNA floating in its environment (and can now use that genetic information!)

Avery, MacLeod, and McCarty discovery and method

• These three scientists continued Griffith’s work to prove DNA_____ was the chemical passed during transformation

• Mixed the contents of the pathogenic bacterial cells with different enzymes

  • protein______________-destroying enzyme

  • an RNA____-destroying enzyme or  

  • DNA____-destroying enzyme

• Each extract was mixed with live non-pathogenic bacteria to see which molecule was necessary in order for transformation to occur

Found: the only bacteria that was not transformed (remained non-pathogenic) was the one in the extract with the DNA-destroying enzyme

Conclusion: DNA is the genetic material

Chargaff discovery and method

• Showed that nucleotides were present in characteristic compositions:

  • Approximately same amounts of A & T

  • Approximately same amounts of C & G

• Created “Chargaff’s Rule”

• Important because this helped scientists realize that certain bases always pair with each other

Hershey and Chase discovery and method

• They used radioactive isotopes* to trace sulfur and phosphorus in bacteriophages in order to prove that genetic information is carried by DNA and not proteins

• Two different types of Bacteriophages were created:

  • One with radioactive sulphur(found in proteins creating the capsid)

  • One with radioactive phosphorous (found in DNA contained in the capsid) 

• In two different experiments the viruses infected bacterial cells (one had radioactive sulfur, one had radioactive phosphorous)

• Remaining protein shell was shaken from the outside of the bacterial cell

Centrifugation was used to separate the liquid around the bacterial cells from the bacteriophages in order to examine what was__injected

Watson and Crick discovery and method

Used information from other scientist’s discoveries to successfully create a model of DNA and therefore discover the structure of the DNA molecule → Chargaff’s rule and Franklin’s Photograph 51

Wilkins discovery and method

Franklin discovery and method

• Used X-Ray diffraction (shining X-rays on crystals of DNA molecules) to examine and show patterns on photographic film. 

• Worked in the same lab as scientist Maurice Wilkins

• Franklin obtained a high resolution photograph of DNA using X-ray crystallography (photo 51)

  • Was able to conclude that DNA had a helical structure

    • Determined that 0.34 nm separated adjacent base pairs

    • Determined that one turn of the helix is 10 base pairs (3.4 nm)

Also realized that the sugar-phosphate backbone was located on the outside of the helix, while the nitrogenous bases were on the inside

Similarities between prokaryotes and eukaryotes

• Require origins of replication

• Have elongation occur in the 5’ to 3’ direction

• Have continuous synthesis of a leading strand and discontinuous synthesis of a lagging strand

• Require the use of a primer for synthesis of the leading strand and for each of the Okazaki fragments on the lagging strand

• Use DNA polymerase enzymes

Differences between prokaryotes and eukaryotes

The rate of replication in prokaryotes is much faster

• Eukaryotic cells have more elaborate replication machinery (more “subunits” of DNA polymerases involved)

• The presence of histones slows down the process.

DNA replication happens in the nucleus in eukaryotes and in the cytoplasm in prokaryotes

The linear nature of eukaryotic chromosomes requires telomeres at the ends

Plasmid

a small, circular, double-stranded DNA molecule that is physically distinct from a cell's chromosomal DNA.

Levels of organization of genetic material in eukaryotes

Double helix, nucleosome, chromatin, 30 nm fibres, chromosomes

Stages of DNA replication

Initiation, Elongation, Termination

Initiation

Starts at the origin of replication

DNA helicase enzyme unzips the double helix

Once the enzyme has opened the molecule an unwound oval-shaped area known as the replication bubble forms. 

SSBs bind to the open DNA strands to prevent them from annealing back together 

 topoisomerase II (also called DNA gyrase) relieves the tension brought about by the unwinding of the DNA strands.

Elongation

 the synthesis of the new DNA strands by joining individual nucleotides together based on the parent strand sequence

• To create the new chain energy is needed → this comes from the incoming nucleoside triphosphate  

  • Nucleoside triphosphate: 

    • Nucleoside: sugar + base

    • Triphosphate: 3 phosphate groups

DNA polymerase III cleaves off two phosphates from the nucleoside triphosphate, releasing energy, and the resulting nucleotide is added to the DNA strand, creating the alternating sugar-phosphate backbone.

Replication on the leading strand

• Once the RNA primer is in place, DNA polymerase III can attach and start adding the DNA nucleotides towards the replication fork

• The RNA primer will be removed later and replaced with deoxyribonucleotides (DNA nucleotides)

DNA replication on the lagging strand

• Just like the leading strand, RNA primers are used to build the lagging strand

  • Once the RNA primers are in place DNA polymerase III can add nucleotides to the free 3’ hydroxyl end away from the replication fork

• The RNA primers will be removed later and replaced with deoxyribonucleotides (DNA nucleotides)

• Because fragments extend away from the replication fork, as they lengthen, new primers must be added as replication proceeds

→ Multiple RNA primers are needed for the lagging strand

• Newly synthesized DNA fragments are called Okazaki fragments

Termination

• The protein-DNA complex at each replication fork carrying out replication are referred to as the replication machine

• Termination happens when completed daughter DNA molecules separate from each other

• At this point, the replication machine is dismantled