replication fork
region at which replication is occurring
DNA helicase
unwinds DNA strands, beginning at origins
DNA polymerase requires…
dNTPs and a primer (usually RNA)
DNA topoisomerase
removes supercoils and reduces torsional stress caused by local unwinding
primase
specialised RNA polymerase that forms a short RNA molecule complementary to a single-stranded region of unwound duplex DNA
leading strand
continuous replication in 5’ to 3’ direction by DNA polymerase following the movement of replication fork
lagging strand
discontinuous replication in opposite direction from movement of replication fork
lagging strand replication process
- DNA elongated from RNA primer by DNA polymerase
- Okazaki fragments formed: short, discontinuous fragments consisting of DNA and RNA
- RNA part of Okazaki fragments replaced by DNA
- 2 adjoining DNA molecules ligated together by DNA ligase
replisome
molecular machinery that carries out DNA replication
components of the replisome
- CMG helicase
- Replication Protein A (RPA)
- DNA Pol Epsilon
- PCNA
- Pol Alpha - Primase complex
- Pol Delta - PCNA complex
- RFC - PCNA complex
- Ribonuclease H and FEN-1
- Pol Delta
CMG Helicase
- binds to leading strand
- unwinds parent strands at replication fork through ATP hydrolysis
- contains accessory subunits Cdc45 and GINS complex: inactive without them
Replication Protein A (RPA)
- binds single-stranded DNA to keep its template in optimal conformation for DNA polymerase (Pol Delta)
- prevents formation of secondary structures on single-stranded DNA
DNA Pol Epsilon
carries out leading strand DNA synthesis, forming a complex with PCNA
PCNA
- prevents Pol Epsilon/Delta from dissociating from the template
- homotrimeric protein (made of 3 monomers)
Pol Alpha - Primase complex
- primase forms RNA component of primer
- Pol Alpha extends primer with DNA
Pol Delta - PCNA complex
- on the lagging strand
- replaces Pol Alpha - Primase complex and completes synthesis of Okazaki fragments
RFC - PCNA complex
RFC (Replication Factor C) is PCNA loader: opens PCNA ring and loads it at primer on DNA
Ribonuclease H and FEN-1
displace RNA components at 5’ ends of Okazaki fragments
Pol Delta
replaces RNA with DNA
Origin Replication Complex (ORC)
6 subunit protein that marks replication origins and load helices, tightly regulating DNA replication in eukaryote
MCM helicase activation/deactivation
- inactive form when loaded in G1 phase of cell cycle
- activated in S phase of cell cycle through phosphorylation + interactions with other proteins
DNA replication process
- unwinding of DNA by CMG helicase driven by ATP hydrolysis
- RPA binds to single-stranded regions
- Pol Alpha - Primase complex synthesises primer sequences through base pairing
- Pol Epsilon - RFC - PCNA complexes replace Pol Alpha complexes and extend primer sequences
- further unwinding and binding of RPA gives rise to 2 replication forks
- Pol Epsilon - RFC - PCNA complexes continue to synthesise leading strands
- Pol Alpha - Primasse complexes form primers for lagging strand synthesis
- primer extension + Pol Alpha-Primase complexe replacement done by Pol Epsilon - RFC - PCNA complexes
- primer removal by FEN-1 and ribonuclease H
- strands ligated together by DNA ligase
DNA replication requires… (3)
- a single stranded DNA sequence
- a primer base paired to template with a free OH group
- a source of dNTPs
DNA polymerase
extends the primer to form a new nucleotide: Pol Alpha does initial elongation, followed by Pol Delta
