DNA Structure and Function

DNA Structure

• DNA, or deoxyribonucleic acid, contains the genetic code
• James Watson, Francis Crick, and Rosalind Franklin are credited with discovering the structure of DNA

Nucleotides

• Nucleotides make up the building blocks of DNA
• Each nucleotide contains a sugar (deoxyribose), a phosphate(PO₄-²), and a nitrogenous base (A, T, C, or G)
• A strand of DNA comprises many nucleotides, making it a polymer

DNA Strands

• The DNA structure has 2 strands
• A nitrogenous base can be adenine, thymine, guanine, or cytosine
• Adenine pairs with thymine, while guanine pairs with cytosine
• Purines (adenine and guanine) have a two-ring structure, and pyrimidines (thymine and cytosine) have a one-ring structure
• There is a constant diameter because a purine always pairs with a pyrimidine
• If the base is known on one strand, the other strand can be automatically determined
• The bases connect via hydrogen bonds in the middle of the structure
• This accurate replication is crucial for DNA structure
• The 2 DNA strands are antiparallel, thus reading in opposite directions
• The ends of each strand are designated as the 3' or 5' end, based on the carbon numbering on the sugar molecule
• The 3' end of one strand aligns with the 5' end of the other
• The strands twist into a double helix, facilitated by the sugar-phosphate backbone
• The major and minor grooves allow DNA-binding proteins to recognize base sequences without opening the DNA
• These grooves enable proteins to bind in the correct location

Discovery of DNA as Genetic Material

• Three key experiments helped to identify DNA as the genetic material
• Frederick Griffith (1928): discovered a "transforming factor"
• Oswald Avery, Colin MacLeod, and Maclyn McCarty (1944): transforming factor was DNA
• Alfred Hershey and Martha Chase (1952): confirmed DNA as the hereditary molecule using bacteriophages

Griffith's Experiment

• Griffith used two strains of Pneumococcus: rough (R) and smooth (S)
• The R strain is non-pathogenic, while the S strain is pathogenic
• The S strain produces a capsule due to a mutation in a surface polysaccharide
• There are four types (I-IV), that each elicit a different immune response
• Griffith found that a "transforming factor" converted RII bacteria to an SIII type
• This process was then responsible for heredity

Avery's Experiment

• By using extracts from heat-killed SIII bacteria, Avery determined that the transforming factor is DNA

Hershey and Chase Experiment

• Hershey and Chase confirmed that DNA is the hereditary molecule by experimenting with bacteriophages
• They found that DNA, not protein, is the genetic material passed on to progeny phages

DNA Replication

• DNA replication occurs during cell division, when new cells require a copy of the genetic material

Semi-Conservative Replication

• During semi-conservative replication, each strand of the original DNA molecule remains intact
• Each strand acts as a template for synthesizing a new, complementary copy
• The two resulting daughter strands are identical, each containing one "old" strand and one newly made strand

Bidirectional Replication

• DNA replication is bidirectional; i.e. bacteria have a single origin of replication
• The origin of replication is where the DNA helix unwinds to begin replication, creating a replication bubble
• Replication forks are located at the ends of the replication bubble, where nucleotides are added to newly-synthesized strands
• Eukaryotic chromosomes have multiple origins of replication
• The synthesis begins at multiple origins that merge creating two double-stranded, identical DNA molecules

Replication Machinery

• Origins of replication have consensus sequences recognized by the replication machinery
• These sequences vary between organisms
• The protein complex called replisome carries out DNA replication
• The replisome includes: helicase, topoisomerase, single-strand binding proteins (SSBP), primase, DNA polymerase, sliding clamp, and ligase
• Two replisomes form, moving along the DNA in opposite directions, copying 100 nucleotides per second in humans

Steps in DNA Replication

• Helicase breaks hydrogen bonds between bases, "unzipping" the helix and creating a replication bubble at each replication fork
• Topoisomerase makes one (Topo I) or two (Topo II) cuts in the DNA backbone, allowing the DNA to rotate and relieving torsional stress
• Single-strand binding proteins (SSBP) bind to the newly separated DNA strands, preventing hydrogen bonds from re-forming
• Primase, an RNA polymerase, builds the 5'-10 bp RNA primer to start new strands, as DNA polymerase can only add nucleotides to pre-existing nucleotides
• DNA polymerase III adds nucleotides to the 3' end of a growing DNA strand, synthesizing strands in the 5' to 3' direction, which creates a problem for strands whose 3' end faces away from the replication fork
• Leading strands have continuous synthesis toward the replication fork
• Okazaki fragments have lagging strands, where discontinuous synthesis occurs away from replication fork
• The DNA polymerase I replaces RNA primers with DNA nucleotides, one at a time
• DNA polymerase I has two enzymatic domains: exonuclease (removes RNA nucleotides) and DNA polymerase (adds DNA nucleotides in their place)
• DNA ligase creates a phosphodiester bond to connect Okazaki fragments, after the last nucleotide from the RNA primer has been replaced with DNA

DNA Polymerase III

• DNA Polymerase III is a holoenzyme, enzyme with a core catalytic subunit plus additional proteins DNA Pol III holoenzyme is composed of 2 core (pol III) enzymes linked to a clamp loader + sliding clamp
• This holoenzyme increases the processivity enzyme catalyzes many reactions with a single substrate binding event

Proofreading and Telomeres

• DNA Pol III proofreads its work
• If the wrong base is added, no hydrogen bonds will form
• A daughter (new) strand "flips" out to another site on DNA pol III, where 3’-5’ exonuclease’s activity removes several nucleotides
• Telomeres are repetitive DNA sequences at the ends of chromosomes that can't be replaced with DNA
• Leads to shorter telomeres with each cell cycle
• Telomerase extends telomeres using its own RNA template

Telemorase Enzymes

• Elizabeth Blackburn and Carol Greider discovered how chromosomes are protected by telomeres and the enzyme telomerase
• Cells can sense telomere length
• Differentiated somatic cells have low telomerase activity, leading to cells stopping to divide, "senescence," and limited lifespan
• The telomerase activity of germ cells and stem cells, which are "immortal," can divide an unlimited number of times

Cancer and Telemorase

• Telomerase and Disease is the disease that insufficient telomerase activity can cause premature aging (progeria)
• Turning on telomerase in normal cells does not prevent aging
• Increased telomerase activity is common in cancer cells

Molecular Techniques: PCR

• Rapidly amplifies specific DNA sequences from small amounts
• Used extensively in research, forensics, genetic testing, and diagnostics

Components of PCR

• Buffer for correct salt concentration, pH, and Mg2+ levels
• Template for the DNA to be amplified
• RNA primers specific to the sequence
• Deoxynucleotides (dATP, dCTP, dGTP, dTTP) like normal DNA replication
• Taq polymerase from Thermus aquaticus canfunction at high temperatures

PCR Process

• Step 1: Denaturation- heat at 95°C to separate DNA strands The heat breaks the hydrogen bonds
• Step 2. Annealing- cool to 45-60°C to allow primers to bind to template DNA
• Primers will be complementary to the ends of the template strands
• Step 3. Extension at 72°C, Taq polymerase uses primers to synthesize new DNA strands, using dNTPs

Features of PCR

• DNA doubles in each round
• Output is 2" molecules (n=number of rounds)

Electrophoresis

• Load DNA (PCR products) into gel of agarose
• Apply electricity(DNA is negatively charged, runs toward (+) end)
• Smaller fragments move faster
• Stain with the use of DNA-binding dye

Application of a PCR

• Variable number tandem repeats are VNTRs
• Short, repetitive sequences
• Different alleles have numbers of repeats, making different bands on the gel
• Many alleles exist with different numbers
• Each individual has a different band on combinations, create the genetic fingerprint

VNTR's

• Variable area inherited like other alleles
• Runnette squares- used to look at allele combinations for kids

PCR for Diagnosing Infectious Disease

• Extract DNA from Patient sample
• Then perform PCR with-pathogen
• Specific primers
• Look to see a band shows up on the get.

DNA sequencing: Fred Sanger

• The 1980 nobel prize in which used Walter Gilbert,
• The Nobel Prize from Paul Berg" fundamental biochemistry and nucleic acids"

Sanger sequencing: ddNTPs

• Have Carbon
• Nucleotides, these bases at each number from -
• Stop site for enzyme

Sanger Sequencing: Chain Termination

• Incorporated the polymerase at chain and termination

Sanger Sequencing Reaction

• PCR components includes dNTPs

Sanger Technique

• Set up 4, many short fragments on these sections
• The sequencing contains all nucleotides

Automated sequencing

• Used to determine nucleotide
• Automated have peak