Unit 3: Nucleotides and Nucleic Acids, DNA Replication

What are the three components of a nucleotide?

Nitrogenous base, Pentose sugar (ribose or deoxyribose), Phosphate group(s)

What is the difference between a nucleoside and a nucleotide?

Nucleoside = base + sugar; Nucleotide = base + sugar + phosphate(s)

Where does the phosphate group attach on the sugar?

Typically at the 5' carbon of the sugar

Where does the nitrogenous base attach?

At the 1' carbon via a β-N-glycosidic bond

Which bases are purines?

Adenine (A), Guanine (G) → 2-ring structure

Which bases are pyrimidines?

Cytosine (C), Thymine (T), Uracil (U) → 1-ring structure

What is the key structural difference between purines and pyrimidines?

Purines = larger, double-ring; Pyrimidines = smaller, single-ring

Why is purine-pyrimidine pairing important?

Maintains constant DNA helix width and prevents structural distortion

What type of bond connects nucleotides in a strand?

Phosphodiester bond (between 3' OH and 5' phosphate)

In what direction is DNA/RNA synthesized?

5' → 3' direction

What functional groups are required for phosphodiester bond formation?

3' OH (existing strand) and 5' triphosphate (incoming nucleotide)

What is the key difference between DNA and RNA sugar?

DNA: deoxyribose (H at 2'); RNA: ribose (OH at 2')

What must you include when drawing a strand?

Correct sugar numbering (1'-5'), Base attached at 1', Phosphate at 5', Phosphodiester linkage (3'-5'), Directionality (5' → 3')

What is DNA melting?

Separation of double-stranded DNA into single strands due to heat or chemical disruption

What is melting temperature (Tm)?

Temperature at which 50% of DNA is denatured

What increases DNA melting temperature?

High GC content, Longer DNA length, High salt concentration

Why does GC content increase stability?

More hydrogen bonds + stronger base stacking interactions

What happens to absorbance during DNA melting?

Increases (hyperchromic effect)

What does semi-conservative replication mean?

Each daughter DNA molecule contains 1 parental strand and 1 newly synthesized strand

Why is semi-conservative replication important?

Ensures accurate genetic information transfer

What does 'read up / write down' mean?

DNA polymerase reads template 3' → 5' and synthesizes new strand 5' → 3'

Why can DNA only be synthesized 5' → 3'?

Requires free 3' OH for nucleophilic attack

What is the key chemical reaction in DNA synthesis?

Nucleophilic attack by 3' OH on α-phosphate of incoming dNTP

What provides energy for DNA synthesis?

Cleavage of dNTP → dNMP + pyrophosphate (PPi)

What happens to pyrophosphate (PPi)?

Hydrolyzed → drives reaction forward (irreversible)

Why is Mg²⁺ important?

Stabilizes negative charges and assists catalysis

What enzyme unwinds DNA?

Helicase

What prevents DNA strands from reannealing?

Single-strand binding proteins (SSBs)

What relieves supercoiling tension?

Topoisomerase

What synthesizes RNA primers?

Primase

What is the main DNA synthesis enzyme in prokaryotes?

DNA Polymerase III

Which enzyme removes RNA primers?

DNA Polymerase I

What enzyme seals nicks in DNA?

DNA ligase

What is the difference between leading and lagging strand?

Leading: continuous; Lagging: discontinuous (Okazaki fragments)

What ensures correct nucleotide incorporation?

Active site geometry + base pairing

What happens if incorrect base pairs try to bind?

Poor fit → no catalysis

What is the role of induced fit?

Enzyme changes shape to verify correct base pairing

How does proofreading work?

3' → 5' exonuclease activity removes incorrect nucleotides

Why is geometry more important than hydrogen bonding alone?

Shape ensures high fidelity even if H-bonds could form incorrectly

What can you infer from a DNA sample with a high melting temperature?

High GC content and/or longer strand length

What happens if DNA polymerase loses proofreading ability?

Increased mutation rate

Why can't DNA polymerase start synthesis on its own?

Requires a free 3' OH → needs primer

What would happen if ligase didn't function?

Okazaki fragments remain unjoined → fragmented DNA

Why is the lagging strand discontinuous?

Polymerase can only synthesize 5' → 3', opposite of fork movement