cell and molecular lecture 7: DNA replication

DNA must also be copied

- before a cell divides, it must replicate its DNA, so each daughter cell receives a complete copy of the genome

DNA replication begins at an origin

- DNA replication begins at specific regions called origins of replication (ORI)

- MCM helicase unwinds the DNA double helix, creating two replication forks

- replication proceeds bidirectionally from the origin

single-stranded DNA must be stabilized

- RPA (replication protein A) binds exposed single-stranded DNA

- RPA prevents the DNA strands from reannealing and protects the exposed DNA

DNA polymerase cannot start a new strand

- RNA polymerase can begin synthesis de novo

- DNA polymerase can only add nucleotides to an existing 3'-OH, DNA synthesis requires a primer

primase provides the starting point

- primase synthesizes a short RNA primer complementary to the DNA template

- the primer provides the 3'-OH required for DNA synthesis

- DNA polymerase α (Pol α) extends the RNA primer with a short stretch of DNA

the two strands are replicated differently

- leading strand: synthesized continuously toward the replication fork

- lagging strand: synthesized discontinuously away from the replication fork as Okazaki fragments

- both strands are synthesized 5' -> 3'

different polymerases copy the two strands

- Pol ε synthesizes the leading strand

- Pol δ synthesizes most of the lagging strand

- both polymerases synthesize DNA 5' -> 3'

PCNA keeps DNA polymerase on the DNA

- PCNA: sliding clamp that increases polymerase processivity

- RFC: clamp loader that places PCNA onto DNA

- PCNA holds DNA polymerase on the template, allowing continuous DNA synthesis without dissociation

the lagging strand must be completed

- each Okazaki fragment begins with an RNA primer

- remove the RNA primer: RNase H removes most of the RNA primer

- replace RNA with DNA: DNA polymerase fills the resulting gap with DNA

- seal the remaining nick: DNA ligase forms the final phosphodiester bond, joining adjacent DNA fragments

DNA polymerase proofreads as it synthesizes

- DNA polymerase occasionally incorporates the wrong nucleotide

- detect the mismatch: incorrect base pairing stalls DNA synthesis

- remove the incorrect nucleotide: DNA polymerase uses its 3' -> 5' exonuclease activity to remove the mismatched nucleotide

- resume DNA synthesis: DNA polymerase returns to 5' -> 3' synthesis and inserts the correct nucleotide

- DNA synthesis: 5' -> 3' | Proofreading: 3' -> 5'

DNA replication: the big picture

- open the DNA: MCM helicase + topoisomerase + RPA

- start synthesis: primase + pol α

- copy the DNA: pol ε + pol δ + PCNA 5' -> 3' synthesis

- finish the lagging strand: RNase H + DNA polymerase + ligase

- maintain accuracy: 3' -> 5' exonuclease activity

- each parental strand serves as a template for synthesis of a new complementary strand