lec21 Flashcards
3 hypothesized replication methods
- conservative
- semi-conservative
- dispersive
What are the results of the Meselson-Stahl experiment?
Grew bacteria in 15N (heavy) medium → DNA all 15N/15N (bottom band).
Switched to 14N (light) medium:
Generation 1: DNA 15N/14N (middle band).
Generation 2: DNA 15N/14N (middle) + 14N/14N (top, lightest band).
Proved DNA replication is semiconservative.
What are the substrates and building blocks of DNA replication?
Substrates: Deoxynucleoside triphosphates (dNTPs).
Building blocks: Deoxynucleoside monophosphates (dNMPs) incorporated into the DNA strand.
What are the essential components required for DNA synthesis?
- DNA template – Provides the sequence for complementary base pairing.
- dNTPs (deoxynucleoside triphosphates) – Building blocks for DNA synthesis.
- Primer (DNA or RNA) – Provides a free 3’-OH for DNA polymerase.
- Mg²⁺ ions – Essential cofactor for DNA polymerase. positions and stabilizes negative charges
- DNA polymerase – Catalyzes (large protein complex that includes enzyme) nucleotide addition to the 3’ end. proofreads,
How does DNA polymerase extend the growing DNA strand?
DNA polymerase adds nucleotides to the 3’ end of the growing DNA chain by catalyzing a nucleophilic attack of the 3’-OH on the α-phosphate of an incoming dNTP, releasing pyrophosphate (PPi)
What are the roles of cofactor and which in DNA synthesis?
-Stabilize negative charges on dNTPs.
-Assist in deprotonation of the 3’-OH, making it a stronger nucleophile.
-Position reactants for efficient catalysis.
-Facilitate pyrophosphate, P₂O₇⁴⁻, release, driving the reaction forward.
How does the DNA polymerase active site ensure correct base pairing?
The shape of the active site facilitates correct Watson–Crick base pairing. Correct base pairs fit precisely into the active site, while mismatched bases do not, reducing errors during DNA replication.
What does DNA polymerase do when building and fixing DNA?
Building (Polymerizing): Adds new pieces to the DNA train track in the 5’ to 3’ direction.
Fixing (Exonuclease): Erases mistakes from the ends of the DNA.
Can erase forward (5’ → 3’) or backward (3’ → 5’).
whats the one exception to exonuclease function
E. coli DNA polymerase I Klenow fragment:
Builds DNA: Yes (5’ → 3’).
Erases backward: Yes (3’ → 5’).
Erases forward: No (5’ → 3’).
normal can erase in both directions
How does DNA synthesis occur simultaneously on both parent strands?
DNA synthesis happens at replication forks in the 5’ → 3’ direction for both strands. The leading strand is synthesized continuously, while the lagging strand is synthesized in short Okazaki fragments.
What is the key difference between leading and lagging strand synthesis?
- Leading strand is synthesized continuously in the same direction as the replication fork. only one rna primer
Lagging strand is synthesized discontinuously in short Okazaki fragments, requiring RNA primers and looping for coordinated synthesis.
e coli replication
E. coli has a circular DNA map with one oriC (starting “X”).
Helicase unzips it, making two replication forks moving both ways—bidirectional!
DNA polymerase III builds at each fork:
Leading strand: One long piece.
Lagging strand: Short Okazaki fragments with RNA primers.
Is this for all cells? No! It’s special for E. coli and some bacteria. Other cells (like ours) can be bidirectional too, but use different enzymes and rules!
Unidirectional and bidirectional replication are two possible
mechanisms for DNA replication from a single origin t or f
t
Where does prokaryotic DNA replication initiate, and what are its key sequence elements?
Prokaryotic replication starts at oriC, which contains:
Three repeats of a conserved 13-bp A-T rich sequence
Four repeats of a conserved 9-bp A-T rich sequence (sequences of 9 b repeats 3 times)
These sequences facilitate DNA unwinding.
