8.3 DNA Replication

Structure of DNA

• made of two strands of nucleotides

  • Covalent bonds link nucleotides in strand

  • Hydrogen bonds hold two strands together (nucleotides)

Nucleotide and Strand Asymmetry

• The 5’ carbon atom of the sugar is where the phosphate group is attached (5’-P end)

• The 3’ carbon atom of the sugar has a hydroxyl group (3’ -OH end)

DNA Is a Double Helix of Anti-Parallel Strands

• One Strand: 5’➞3’

• Other Strand: 3’➞5’

• Sugar phosphate “backbones”

• Bases “glue” strands together with H-bonds – A=T, G≡C

DNA Replication - Initiation

• Replication forks are built through a process called “licensing” (preRC - prereplication comple

• ORC (origin of replication complex)

• DNA Helicase unwinds double-stranded DNA, but overwinds the DNA in front of it, requiring the action of Topoisomerase to reduce strain

DNA Replication - Elongation

• New nucleotides come in as nucleotide triphosphates (like ATP)

• The loss of two phosphate groups (pyrophosphate) releases energy to power the connection of the nucleotide to the strand

• DNA Polymerases can add new nucleotides to the 3’ end of a DNA strand

• DNA polymerases can “proofread” = replace an incorrect nucleotide just placed

• Requires 3’-5’ exonuclease capability (cut off 3’ end)

  • Exonuclease = Cuts nucleotides off end of nucleic acid strand

• Only adding new nucleotides to 3’ end of strand allows for proofreading and removal of incorrect nucleotides

DNA Replication - Termination

converging replication forks meet, synthesis finishes, replisome machinery unloads, and newly duplicated DNA molecules separate

First step of DNA replication

Unpack the chromatin so that the replication machinery can find and access important DNA sequences

Origin of replication

specific sites (sequences) where DNA replication starts
ex: E. coli : one, Eukaryotes: many (faster for larger genomes

Replication Bubble

expanding area of replicated DNA

Replication Fork

site of active replication, two per replication bubble

Limitations of DNA Polymerases

1. They cannot unwind double-stranded DNA

2. They cannot start a chain, they can only add a nucleotide to an existing nucleotide strand

3. They can only add nucleotides to 3’ ends (5’-3’ synthesis)

4. They cannot link existing DNA chains to each other

To which primerwill DNA Pol start to build a new chain in the direction towards the replication fork?

A - 3’ Top

Leading strand

• Synthesis occurs continuously

• Synthesized towards the replication fork

Lagging strand

• Synthesized away from the replication fork

• series of segments (Synthesis discontinuously), Okazaki fragments

• oldest DNA at replication origin

Okazaki fragments

Discontinuous strands of DNA being replicated on lagging strand

DNA Ligase

• connects adjacent strands of DNA together to combine Okazaki fragments to form one continuous new strand

• “Ligate DNA” means to join two existing DNA sequences into a single string

End Replication Problem

• Eukaryotic linear chromosomes can never be fully replicated due to lagging-strand dynamics

• Chromosomes shorten during each replicative cycle

Telomeres

• non-protein coding repetitive sequence found at the ends of linear chromosomes

• Act as a buffer to protect protein-coding genes

• Shorten with each replication

• Limit replicative potential of cells —> prevents cancer, but may contribute to aging

In which cells would you not want telomeres to shorten?
A) Neural cells
B) Germline cells
C) Pluripotent stem cells
D) Terminally differentiated cells

B) Germline cells

Insulin production

• Use PCR to amplify the insulin gene

• Use Restriction Enzymes to make compatible sticky ends of plasmid DNA and PCR product

• Use ligase to glue the pieces together