Gene-L6-DNA replication I

what are the key features and requirements of DNA replication in human cells?

• must be highly efficient as large genome and high fidelity as error can cause major effects especially in coding regions

• each parental strand serves as a template for a new complementary strands

• DNA strands are antiparalellel

what are the major challenges of DNA replication in eukaryotic cells?

1. timing and co-ordination

• entire genome must be replicated once per S phase

• multiple origins of replication must be synchronised

2. speed

• large genome requires rapid replication within a limited time

3. accruacy

• must maintain genome integrity and errors cause mutations

what does it mean that DNA is semi conservative

• eat parental DNA strand is used as a template for a new strand

• so daughter DNA molecules have 1 original parental strand and 1 newly synthesised strand

• half old half new

what are the features of replication? how were these identified?

1. semi conservative

2. begins in the replication origin

3. occurs bidirectionally

found in bacteria- E.coli studies

what are the 3 major structural challenges of DNA replication and how are they overcome?

1. DNA is a double helix- must be unwound and strands must be separated at replication origins

2. DNA polymerase cannott start synthesis de novo- needs a primer with a free 3’ OH group

3. DNA is antiparallel by synthesis is 5’ to 3’. leading strand is continuous and lagging strand is synthesised discontinuously Okazaki fragments

why does DNA polymerase require a primer? how is it made and removed?

• needs a primer with a free existing e’ OH group

• primase RNA polymerase synthesises a short RNA primer of about 5 nucleotides

• primer is complementary to template DNA

• DNA polymerase extends this primer with DNA

• primer is then removed by hydrolysis and replaced with DNA

how are RNA primers removed and replaced during bacteria DNA replication?

• DNA pol III synthesises DNA intiially

• at primer- polymerase switching occurs to DNA polymerase I

• DNA pol I- has 5’ to 3’ exonuclease activity and removes RNA primer nucleotides and replaces them with DNA

• remaining gaps are sealed by DNA ligase

how do leading and lagging strands synthesis differ during DNA replication?

leading strand

• synthesised continously

• requires only one RNA primer

lagging strand

• synthesised in fragments- okazaki

• requires multiple RNA primers

• each fragment: primer added and extended by DNA polymerase. then primer is removed and replaced with DNA. fragments are joined by DNA ligase

what are the main enzymes and accessory proteins at the replication fork? what are their functions? 6

1. heliacse- uses ATP to unwind helix

2. primase- RNA polymerase to make RNA primers

3. SSB- single stranded binding proteins- prevent reannealing of DNA strands and stabilise ss DNA

4. topiosomerase- relieves supercoiling and torsional stress and cuts/joins phophosdiester bonds

5. DNA pol- 5’ to 3’ and have exonuclease proofreading activity

6. DNA ligase- joins Okazaki fragments

key steps in initiation of DNA replication at origin site?

• replicate begins at conserved origins of replication

• origin binding proteins recognise specific sequences

• helices is loaded onto DNA and uses ATP to separate strands

• primase synthesises RNA primer

• forms the initial replication fork for DNA synthesis

structure of OriC origin in E.coli? how do specific sequences function in initiation?

• OriC is 240bp

• has 4 × 9bp repeats for DnaA to bind- to separate DNA strands

• 3 × 13bp AT rich repeats- easier to unwind. binding site DnaB helicase forms pre priming complex

• DnaG- primase binds to initiate primer synthesis

major differences between DNA replication I prokaryotes vs eukaryotes?

• ecoli

• 4m bp genome and single circular chromosome and have 1 single origin of replication OriC

• humans

• 6m bp genome and 23 linear chromosomes. need 30k origins of replication. replication is slower and more complex

how is DNA replication initiated in eukaryotic cells at a molecular level? what are these called?

1. origins are AT rich sequences recognised by origin binding proteins

2. pre-replication complex formation: MCM helicase and CDC6 and CDT1(helices loading factors)- load and stabilise helices onto DNA

3. these proteins act as licensing factors and ensure origins are prepared for replication

origins: located near promotors and transcriptionally active regions

these are called licensing factors

what ensures that DNA replication is initiated from each replication origin during S phase? what prevents re-initiation of DNA replication at origins that have already been used?

• pre replication complexes assemble at origins during G1 phase with key licensing factors Cdc6, cdt1, MCM helicase

• during S phase: cyclin CDK complexes phosphorylation pre-replication complex components and activates origins and initiates DNA replication

• after replication- licensing factors cdc6, cdt1 are degraded to stop helices cant be reloaded and stops the formation of new pre-res and each origin site is used only once per cell cycle

how do cyclin CDK complexes regulate DNA replication?

• cell cycle oscillated low and high DK activity and driven by cyclin synthesis and degradation

• CDKs act by phosphorylating target proteins

• G1: activate transcription of S phase genes

• S phase CDKs initiate DNA replication

• M phase- control chromosome degradation

what are specific cyclin CDK complexes involved in the mammalian cell cycle?

G1: cdk4 and cdk6

late G1: cdk2 E

s phase: cdk2 A

g2/M- cdk1 A and B

regulated by Cdk inhibitor CIP and INK4