Leading & lagging strand DNA synthesis, Okazaki fragment maturation & replication termination

What is used in establishing bidirectional replication?

Replisome comprised of replicative helix (CMG complex, unwinds dsDNA, comprises Cdc45, MCM & GINS) & the leading strand DNA polymerase

What roles do single stranded DNA binding proteins (SSBs) play?

Replication protein A (RPA) facilitates DNA unwinding by trapping exposed ssDNA & protects ssDNA from degradation

What DNA polymerases are present at the eukaryotic replication fork?

Pol ε, Pol α-primase, Pol δ

What is the role of Pol ε?

Synthesises leading strand only

What is the role of Pol α-primase?

Synthesises RNA-DNA primer to prime synthesis of every Okazaki fragment on lagging strand

What is the role of Pol δ?

Synthesises Okazaki fragments on the lagging strand, extending from primers made by Pol α-primase

How is the leading strand primed?

By the first Okazaki fragment on the lagging strand, nascent leading strand made up of DNA synthesised by Pol α-primase, Pol δ, and mostly Pol ε

What are sliding clamps?

• Ring shaped molecules with six fold symmetry

• DNA polymerase processivity factors

How are sliding clamps used in eukaryotes?

PCNA interacts with pol δ & pol ε to tether polymerases to template DNA, resulting in a huge increase in polymerase processivity

How is PCNA loaded onto DNA?

Clamp loader replication facotr C loads PCNA onto DNA, which is a heteropentameric protein complex with each subunit a member of AAA+ family

How is PCNA specifically laoded at 3’ end of RNA-DNA primer?

Through conformation changes brought about by ATP binding & hydrolysis

How are Okazaki fragments processed in eukaryotes?

• Pol δ-PCNA complex envounters previously synthesised Okazaki fragment & performs strand displacement synthesis

• Fen1 cleaves displaced 5’ flap

• DNA ligase I ligates DNA ends to form continuous strand

How do multiple proteins bind to PCNA?

Via a common sequence motif (PIP motif)

When does replication end?

When forks converge

What is the process of replication termination in eukaryotes?

• Forks converge

• Converging replisomes pass each other & move on dsDNA

• Pol δ processes final Okazaki fragments

• Replisome disassembled from replicated DNA using ubiquitylation of MCM & action of p97/Cdc-48 afterwards

• Topoisomerase added to decatenate DNAs