L4: Eukaryotic chromosome replication II

What is the first enzymatic step of DNA replication

1. localised separation 

   • or unwinding of the two DNA strands at the replication origin

   • catalsed by DNA helicases

2. Unwound DNA is stabilised by single-stranded binding proteins

3. DNA polymerases and additional proteins are recruited thhat built up active DNA replication forks

The replicative DNA helicase: the eukaryotic helicase is…

• a core complex of six MCM proteins

• with many other associated proteins:

  • Cdc45

  • GINS complex

1. Loading the MCM complex (the helicase)

1. MCM double hexamer complex is loaded at replication origins in an ATP-dependent manner

   • by origin recognition complex ORC

2. Involving Cdc6 and cdt1 proteins

2. Activation of the MCM helicase in S phase

1. Loaded but inactive MCM double hexamer complex is converted into  an active form

2. With protein kinases CDK and DDK

   • with association with several other proteins:

   • cdc45 and GINS

3. Complex: CMG complex 

   • (Cdc45, MCM, GINS)

3. What does DNA helicase activation lead to

1. local unwinding 

2. separation of the double hexamer complexes

4. Where do each of the two CMG  helicase complexes travel

• travel with one of the two emerging replication forks away  from the initiation site

5. After unwinding, the active CMG DNA helicase…

1. translocates on the DNA leading strand

   • in 3’ to 5’ direction 

2. Dependent on ATP hydrolysis

THEREFORE→ displacing the complementary DNA strand

6. What does this cause?

1. Around activated helicases, functional DNA replication fork complexes are assempled

2. Involving the recruitment of the DNA polymerases and replication factors

Types of DNA polymerases→ 6 major DNA polymerases (pol) in eukaryotes

But, DNA polymerases make mistakes, they are corrected by…

1. Proof-reading exonuclease activity

2. by mismatch repair systems

Error rates of the pol

• pol alpha is the worst

• pol gamma or epsilon

• pol gamma or epsilon 

Replication factors: other crucial proteins of the eukaryotic DNA replication fork include:

1. DNA helicases→ unwind the two DNA strands, generate ssDNA templates

2. RPA→ single strand binding protein, stabilities the unwound strands, recruits pol alpha/primase

3. PCNA→ sliding clamp, binds  to pol gamma and epsilon, Fen-1 and others

4. RFC→ loads and unloads PCNA

5. Fen-1→ flap endonnuclease, removes sort primers

6. Dna2→ endonuclease, removes long primer flaps

7. DNA ligase I→ joins Okazaki fragments

8. DNA topoisomerases→ release superhelical stress

Please note:

• my notes from BoC are so much better than this

On top of this…recent proteomic analysis of isolated DNA replication fork complexes have identified…

• large amounts of additional proteins which play a role in

  1. maintaining replication fork stability

  2. facilitating replication of damaged DNA 

  3. replication of chromatin templates

DNA synthesis at DNA replication forks: concerted action of these core replication proteins during DNA strand synthesis in eukaryotes

1. INitiation and elongation of DNA strand synthesis

   1. → applies to both leading and lagging strand

2. Maturation of Okasaki fragments (lagging strand)

1. INitiation and elongation of DNA strand synthesis

2. Maturation of Okasaki fragments (lagging strand)

To establish a DNA replication fork…

• both leading and lagging strand synthesis are coupled

• the lagging strand is looped back to obtain co-linearity

  • → trombone model

The proteins of the replication fork thus form…

• a complex ‘molecular machine’

DNA topoisomerases: The immense length of DNA in the nucleus generates topological problems…

1. Winding

   •  Average human chromosome (150Mbp)

   • DNA strands wind around each other (1.4 ×107 times)

   • These turns must be removed during replication

2. Supercoils

   • separation of DNA strands during transciption and DNA replication generates positive supercoils

   • ahead of the moving polymerase complexes

   • would eventually pprevent further elongation

How do DNA topoisomerases resolve this issue

• altering the number of times DNA strands wind around each other

How does DNA topoisomerase I work?

1. nicks one strand of a DNA duplex

2. attaches a DNA phosphate group to a tyrosine residue in its active centre

3. covalently forming a new ester bond

4. Allows roatation of the free end of the cut strand around the uncut single-strand

5. seals the nick

   • breaks the ester bond of the DNA

   • with tyrosine

   • re-ligating the DNA without requiring ATP

   • these are trans-esterifications

This process can therefore…

1. Remove strain imposed on a molecule by local helix unwinding

   • as found in front of active DNA or RNA polymerases

HOw does DNA topoisomerase II work?

1. cuts both strand 

2. bridges the gap

3. allwoing other regions of DNA duplex to pass through before

4. resealing, removing supercoils from the DNA

This enzyme can also…

• Separate interlocked DNA rings (concatemers or catenanes)

This property is essential in

• the final stages of DNA replication

• and during mitosis

Replication of telomeres: termination problem

• mechanism of co-ordinated leading and lagging strand synthesis

• leads to loss  of DNA at the linear ends of the chromosomes→ telomers

  • shorted after each cycle

How is this loss counteracted?

1. Enzyme telomerase can elongate the ends by

2. synthesising and adding new telomere repeats onto the ends

3. using own RNA template