Cell Biology (Notes 34)

Final Exam

The Johnson and Rao Cell Fusion Experiments (1970)

• A G2 nucleus fuses with an S nucleus to become a heterokaryon that progresses through the cell cycle

• G1 nucleus waits for the S phase nucleus to catch up in the cell cycle —> The S nucleus becomes G2 and then both proceed into M phase at the same time

• The G2 nucleus in the presence of S phase stays in G2 - it DOES NOT RE-ENTER S PHASE

  • Doesn’t take a step backward

  • Why is this not occurring?

  • DNA is only licensed to replicate once per cycle

Replication Begins at Origins of Replication

• Replication begins at the ORIGIN (called the REPLICATION ORIGIN)

• Essentially, the replication origin is a stretch of nucleotides or hundreds of base pairs (A-T)

  • Characteristic of this length of DNA is it’s rich in As and Ts

• This is important because the two strands of DNA have to separate in order for replication of each strand to occur

• It is much easier to break apart AT rich DNA than GC rich DNA because A’s and T’s have two H bonds between them

  • G’s and C’s have three H bonds

  • The cell is set up well to separate DNA

• In different groupings of cells, there are certain nucleotide sequences that serve as recognition site for proteins involved in replication process to bind to DNA

• There is a lot going on in the relatively short DNA sequences — in eukaryotes, there are multiple origin replications on the chromosome

  • Not all of the origins of replication are active - that is replication is not initiated at each origin at the same time

    • Clusters or origins that become active

  • If you look at the distance between the origins of replication along the chromosome

• Not all the origins become active at the same time

  • Euchromatin replicates early in S phase

  • Heterochromatin replicates later in S phase

  • This is due to the amount of DNA compaction in euchromatin (less compaction) vs heterochromatin (more compaction)

ORC - Origin Recognition Complex

• Sits at the origin

• Complex of proteins that is a mark on the chromosomes - the ORC sits there through the entire cell cycle unless it is displaced in the brief period of time when a replication fork moves through this region and DNA replication is occurring

• Have to get the ORC out of the way to let DNA replication to occur

• After replication occurs, ORC rebinds to the origin

• IMPORTANT PROTEIN COMPLEX to allow process to occur once

Licensing for DNA Synthesis Begins in Late M and G1

• When replication is occurring and there’s a strand of DNA and a replication fork moving along, you need to unwind the DNA strand

  • This is done through enzymatic action of helices

• ORC bound to helicase forms the PreRC

• The PreRC stands for the pre-replicative complex

• Big protein complex side-by-side bound to each other at the origin of replication

• As the cell moves into S phase, there’s a Cdk and an S phase cyclin bound to each other

• What will happen is that the helicase will become active

  • This will allow the DNA strands to separate

• There are other proteins that interact with the helices - those are phosphorylated by the S phase Cdk kinase

• In order to activate the helicase, some of those proteins have to be phosphorylated by S phase cdk complexes

• Multiple events are occurring by S-Cdk activation and phosphorylation MUST OCCUR

ORC Inactivation

• Inactivated by Cdk

• When ORC is phosphorylated by Cdk, it can no longer bind to the helicase

• In S phase and replication is occurring, the replication forks move to the left and right

  • Helicase is moving away from the origin

• Towards the end of S phase, there is replicated DNA — going through M phase-Cdk activation

• In Late M phase, there is going to be the assembly of new pre-replicated complexes

• As this cell leaves M phase and enters G1, there will be ORC helicase complexes re-assembling at the origins of replication

• This is the license to be able to replicate the DNA again - in order to say DNA is ready to replicate, the preRC must be re-assembled at the origins of replication

• Already in G2, there are preparing events that are the foundations — in late M phase, early G1, the foundation is being laid for the next round of replication and the next S phase

• The other protein that is important is GEMININ

Geminin

• Protecting the newly replicated DNA from having the complexes REBIND to it

• In M phase that the Gemini is destroyed

• Licensing complexes then bind to the DNA - something is being destroyed

  • Destroyed through APC

  • Ubiquinated and destroyed by proteasome