Replication

Question 1

Semiconservative Replication

Answer 1

During replication, two strands of parental duplex separate (breaking H bonds)

• Each parental strand serves as a template for the synthesis of a new complimentary strand (using bp rules!)
• Daughter DNA will have one parent strand and one new strand

Question 2

Evidence for Semiconservative Replication (Meselson and Stahl)

Answer 2

EXPERIMENT 1:

• E.coli grown for many generations in medium w/ heavy nitrogen isotope (n15)
• E.coli then transferred to medium w/ light nitrogen isotope (n14) so that every new strand of replicated DNA would contain n14 instead of n15
• Density centrifugation analysis: DNA samples extracted and centrifuged, separating DNA based on density (n15 on bottom, 14 on top)

OBSERVATIONS:

1) DNA grown in n15 media had one band
2) DNA after one round of replication in n14 media had intermediate band (higher than n15 band/(1))
Therefore, (2) must contain a hybrid of n14 and n15 DNA. Hence, DNA replication is NOT conservative.

EXPERIMENT 2: is it dispersive or semiconservative?

• Bacteria allowed to grow for many generations in n14 (after being transferred from n15)

OBSERVATIONS:

• DNA found to have n14 band AND
• DNA found to have hybrid

Hence, DNA replication is SEMICONSERVATIVE.

Question 3

Fluorescent Nucleotide Experiments

Answer 3

• Eukaryotic DNA replication tracked
• Rounds of cell division/replication resulted in cells containing hybrid and fully labelled nucelotides (seen by fluorescing strands)
• Further support for semiconservative model of DNA

Question 4

Initiating Replication in Prokaryotes

Answer 4

• Begins in S phase (cell cycle)
• Site: single origin of replication
• Replication continues around the circular chromosome

Question 5

Template and Daughter strand

Answer 5

• Template strand runs 3’ to 5’
• Daughter strand runs 5’ to 3’
• 3’ OH of growing strands attack high-energy phosphate bond of incoming nucleotide, providing energy to drive reaction
• Releases a pyrophosphate, third phosphate used to make bond

Question 6

Replication Forks

Answer 6

Regions with the separation of parental strands caused by the unwinding of DNA double helix.

• Within origins of replication

Question 7

Initiating Replication in Eukaryotes

Answer 7

• Parent strands separate (replication forks)
• RNA primer synthesized and base paired w/ template DNA strands

Question 8

RNA Primer

Answer 8

• Short RNA molecule (5-10 nucleotides)
• Synthesized and base paired w/ template DNA
• Required as DNA polymerase (for elongation) only works w/ existing piece of DNA/RNA

Question 9

Replication Elongation

Answer 9

• Polymerization of daughter strand catalyzed by DNA polymerase, 5’ to 3’

Question 10

DNA Polymerase

Answer 10

Catalyzes the polymerization of daughter strand in 5’ to 3’ direction.

• Synthesizes replicated DNA strand from the RNA primers that anneal to template strand

Question 11

Continous and Discontinous Replication

Answer 11

Occurs as DNA polymerase III can only polymerize in 5’ to 3’ direction.

Leading strand: Replication of one daughter strand is continuous (5’ to 3’)

• Only one RNA primer is required

Lagging strand: Replication of one daughter strand is discontinuous (3’ to 5’)

• Contains Okazaki fragments: Fragments of DNA separated by RNA primers
• DNA polymerase I replaces RNA primers w/ DNA nucleotides
• Delay in synthesis relative to leading strand

Question 12

The Replication Initiation Complex

Involved proteins

Answer 12

• Helicase binds to parental DNA strands at origins of replication and UNWINDS double helix
• Single-stranded binding proteins (SSBs) binds to each parental strand until elongation begins to prevent them from annealing
• Topoisomerase binds upstream of replication fork and minimizes torsional strain brought about unwinding that occurs at replication fork

Question 13

Helicase

Answer 13

• Initiation of replication
• Enzyme that binds to parental DNA strands at origins of replication and UNWINDS double helix
• Breaks H-bonds between complementary nucleotide bp

Question 14

Topoisomerase

Answer 14

• Binds upstream of replication fork and minimizes torsional strain brought about unwinding that occurs at replication fork
• Relives stress of unwinding

Question 14

Single-stranded Binding Proteins (SSBs)

Answer 14

• Stabilizes single strands of DNA
• Prevents from separated strands from annealing

Question 15

The Replication Elongation Complex

Answer 15

• RNA Primase synthesizes RNA primers (allows DNA polymerase to elongate)
• DNA Polymerase III extends the RNA primer, responsible for the elongation of daughter strand
• DNA Polymerase I removes RNA primers and replaces them with DNA nucleotides
• Ligase joins Okazaki fragments together following the removal of RNA primers

^ DNA pol. 3 does elongation work in PROKARYOTES

Question 16

DNA Polymerase I

Answer 16

• Removes RNA primers at the end of replication
• Replacing them w/ DNA nucleotides

Question 17

Difference in Prokaryotic and Eukaryotic DNA Replication

Answer 17

• Eukaryotes use a different set of DNA polymerases

AND

• Prokaryotes have a single origin of replication
• RNA primers are removed and replaced with DNA
• DNA ligase seals the fragments, resulting in a continuous DNA strand with no gaps
• In eukaryotes, gaps can remain after RNA primer removal—especially at the ends of linear chromosomes—because DNA polymerase cannot fully replace the final primer

Question 18

Post-Replication Processing of Lagging Strand

Answer 18

• Replacement of the RNA primer with DNA leaves a nick in the sugar-phosphate backbone, where a 3′-OH and 5′ phosphate are not yet joined
• DNA ligase seals the nick by forming a phosphodiester bond.

Question 19

DNA Ligase

Answer 19

• Catalyzes formation of a phosphodiester bond between 3’ end of Okazaki fragment to an adjacent DNA nucleotide
• Joins adjacent replicated Okazaki fragments together

Question 20

Innate Proofreading Mechanism

Answer 20

• Errors can occur during base pairing between incoming nucleotides and the template strand
• DNA polymerase III proofreads each nucleotide as it is added (3′ → 5′)
• If an incorrect nucleotide is added → it is removed and replaced with the correct one
• Additional enzymes (e.g., mismatch repair proteins) can further correct errors after replication

Question 21

Eukaryotic vs. Prokaryotic Replication

Answer 21

Differences:

• PROK: Begins at ONE origin of replication, after which replication continues around the circular chromosome
• EUK: Begins at many different origins of replication (or ori sites) along linear chromosomes

Common:

• RNA primer
• Elongation in 5’ to 3’
• Leading and lagging strand

Question 22

The Problem w/ Replication of Linear DNA Molecules

Answer 22

• Leading strand replicates the whole template strand
• After removal of the final RNA primer on the lagging strand, DNA polymerase cannot replace it with DNA
• There is no upstream 3’-OH group available for nucleotide addition, leaving the chromosome end unreplicated
• Chromosome becomes slightly shorter each replication cycle

DOES NOT OCCUR IN PROKARYOTES bc they have circular chromosomes that lack free DNA ends, so removal of the final RNA primer does not leave an unreplicated region.

Question 23

Telomeres

Answer 23

• Regions at the ends of linear eukaryotic chromosomes
• Composed of short, repetitive DNA sequences (TTAGGG in humans), creating G-T rich sequences
• Act as protective buffers that prevent loss of coding DNA during replication
• Shorten with each round of DNA replication (therefore shorter in older cells/individuals)
• Maintained by telomerase in certain cell types

Question 24

Exceptions to Telomere Shortening

Answer 24

* Gametes and Stem cells * Shortening of gamete chromosomes could lead to missing information * Shortening of stem cells could restrict development of an organism * Thanks to **telomerase** enzyme

Question 25

Telomerase

Answer 25

Catalyzes the length of telomeres in eukaryotic cells. * Specific type of reverse transcriptase (synthesizes DNA from RNA template) * Ribonucleicprotein that contains RNA template as part of complex itself, allowing elongation of linear chromosomes of stem and gametes * May play a role in aging and cancer progression

Question 26

Process of Telomere Elongation

Answer 26

* Telomerase binds to tail of telomere * Catalyzes extention of template strand by adding telomere repeats * Once elongatino occurs, daughter strand replication can be completed