Nucleic Acids

What is Wilkens and Franklin responsible for?

• X-ray crystallography - diffraction patterns

  • Discovered the pattern of DNA structure

What is Watson and Crick responsible for?

Creation of the double-helix model

• Concluded that DNA had

  • 2 antiparallel sugar-phosphate backbones

  • 3’ and 5’ (left vs. right)

  • Nitrogenous bases are paired in the molecule’s interior

    • Adenine (A) with Thymine (T) - has 2 bonds

    • Cytosine (C) with Guanine (G) - has 3 bonds

What is DNA?

A polymer of nucleotides

• Nucleotide contains

  • A nitrogenous base

  • A sugar

  • A phosphate group

What are purines?

Purines have double rings

• A and G

Match up with a pyrimidine

What are pyrimidines?

Pyrimidines have single rings

• T and C

Match up with a purine

What are the characteristics of the DNA double helix?

Double helix is held together by H-bonds between the bases

• A-T - 2 H bonds

• G-C - 3 H bonds

What did Arthur Kornberg discover?

• How DNA is synthesized - 4 components required

  • Nucleotides (A, C, T, G)

  • DNA template

  • DNA polymerase

  • Mg²⁺ (optimizes DNA polymerase activity)

What are the single nucleotides (dNTPs)?

Deoxyribonucleoside 5’-triphosphate: sugar-base + 3 phosphates

• dATP - adenine

• dTTP - thymine

• dGTP - guanine

• dCTP - cytosine

How does DNA replication work?

Each strand of DNA will act as a template for building a new strand

• DNA replication is semi-conservative

  • Each daughter has 1 parent strand

What are base pairing rules?

• Parent strands unwind — used as a template for a new strand

• Two daughter strands are built from new nucleotides

• 2 new strands have formed

  • Each DNA strand has a parent and a daughter strand

Where does DNA replication begin?

It begins at the origins of replication (sites of origin)

• Eukaryotes have hundreds/thousands of these sites along each chromosome.

  • Parent DNA strands separate at the bubble

  • Daughter DNA strands grow at the replication fork

How are the parent strands read for DNA replication?

From 3’ to 5’ - in order

What direction are the daughter strands built for DNA replication?

From 5’ to 3’

What are the first 4 steps/enzymes of the replication sequence?

1. Use initiator proteins — bind to the replication origin sequence on the DNA; trigger helicase

2. Helicase binds — attaches to initiator proteins; binds/loads onto DNA and unwinds helix

3. Binding proteins — stabilize single template strands to keep them open

4. Primase — primes the strands by synthesizing a short RNA primer; gives a starting point

What enzyme is responsible for enlongating?

DNA polymerase III — elongagte the new strand by adding nucleotides to the 3’ end ONLY

• Requires -OH group (only found on ‘3 end) to attach next piece

  • Build from 5’ - 3’

What are the functions of DNA polymerase I and Ligase in the DNA replication sequence?

• DNA polymerase I — replaces primer RNA with permanent DNA nucleotides

• Ligase — glues the fragments into one continuous strand (connects segments)

What is the difference between the leading and lagging strands?

• Leading strand

  • Synthesizes a daughter strand continuously (uninterrupted)

  • Moves towards the replication fork — where the DNA is unzipped

• Lagging strand

  • Made as a series of segments (disconnected pieces)

    • Formed from Okazaki fragments (individual segments)

    • Fragments joined by DNA ligase into a single strand

  • Moves away from the replication fork — formed towards opposite direction of unzipping DNA

What primers are needed, where and why?

Process is initiated by RNA or DNA primer — DNA polymerase cannot initiate synthesis; can only add nucleotides to existing 3’ ends

• Leading strand — only 1 primer needed at the start; synthesis is continuous

• Lagging strand — every Okazaki fragment must be primed individually to be formed

What are the steps/enzymes of leading strand synthesis?

• Primase adds RNA primer only once — begins the strand

• DNA polymerase III builds continuously towards the fork — same direction of DNA unzipping

• Polymerase I replaces primer — removes starting RNA and swaps with nucleotides

• Ligase binds it to the initial segment — connects strand to the rest of the DNA

What are the steps/enzymes of lagging strand synthesis?

• Primase adds short primer sequence — new starter piece of RNA begins segment

• DNA polymerase III adds nucleotides to 3’ end until reaches next primer (Okazaki segment)

  • Builds backwards in segments

• DNA polymerase I replaces primer nucleotides with DNA — removes temporary RNA

• Ligase bonds segments together — connects individual Okazaki segments into one strand

What is proofreading (mismatched repair)?

Enzymes identify, remove and replace damaged stretches of DNA with proper bases

• It only takes one wrong amino acid to cause a mutation; polypeptide cant fold properly

e.g. DNA structural damage can be caused by sun

What are thymine dimers?

When 2 thymine bases (T) become covalently linked — disrupts genetic code

• Can be caused by UV light exposure

• Hard to break these bonds

What happens to eukaryotic chromosome ends during replication?

Chromosome ends get shorter with each replication — lagging strand machinery cannot copy the end of the strand

• Nucleotide sequences called telomeres postpone the erosion at ends

  • Formed from repeater non-coding sequences of DNA — allows for erosion to occur with replication

  • “Junk DNA”

What is telomerase function and where is it active?

Telomerase adds nucleotides (telomeres) to the ends of the DNA — this prevents loss of true genes with replication during cell division

• It catalyzes the lengthening of telomeres in germ cells — maintains chromosome length in gametes, stem cells, reproductive cells

What is a polymerase chain reaction (PCR) and its steps?

A method of rapidly making many copies (millions) of a piece of DNA — large amounts of DNA can be made from a small starting sample

• DNA is heated to separate the two strands

• DNA is cooled — DNA polymerase (Taq) is used to replicate the strands

  • Bases and primers are added to the mix to give polymerase a starting point

• Cycle can be repeated 30-40x