Week 6 (Lectures 3&4)-The Replicon Model:Replication is controlled by a DNA sequence called a replicator that binds an “Initiator” protein. The initiator protein regulates replication of the DNA. DNA synthesis actually begins at the origin, which is usually within or adjacent to the replicator. The origin of replication is where the primase actually first lays downa sequence. -Main job of initiator is to recognize the sequence that is the origin, basically says “this is where we will start” and begins process of melting or unwinding DNA at the originAll DNA is replicated from a origin of replication known as the replicon(in bacteria). On the other hand, eukaryotic chromosomes have multiple replicons. The replicon was proposed to have two components to control the initiation of replication: The replicator and the initiator. Replicator = the entire set of cis-acting Dna sequences that is sufficient to direct the initiation of DNA replication. Origin of replication is always part of the replicator. -Roles of the Initiator:-Initiator binds DNA.-Initiator unwinds (a little) DNA at the origin.-Initiator recruits other replication proteins.This protein recognizes a DNA element in the replicator and activates the intitation of replication. Initiator proteins have been identified in many different organisms. They elect the sites that will become origins of replication, though they recognize DNA using different DNA-bindingmotifs. It is the only sequence specific DNA-binding protein involved in the initiation of replication. The remaining proteins required for replication intiation don’t bind to a DNA sequence specifically. They are recruited through protein-protein interactions.They always perform three functions: 1.) bind a specific DNA sequence within the replicator. 2.) interact with additional factors required for replication initiation, thus recruiting them to the replicator. 3.) distort or unwind a region of DNA adjacent to binding site in order to facilitate the initial opening of the DNA duplex.-unwinding of DNA provides ssDNA template for additional replication proteins to begin the RNA and DNA synthesis steps of replication. But formation of ssDNA is not sufficient to recruit DNA helicase and other proteins. Initiatior protein itself recruits the proteins, so replication is linked to the control of initiator activity. Genetic identification of replicators:1. Replication origins -have ability to allow a piece of DNA to replicate outside of chromosome. -Episome – a piece of DNA that could live outside the chromosome. The presence of a replication origin on the plasmid allows this2. Selectable marker important for replication -some gene that allows us to ID cells that have picked “this” up ex: ecoli, antibiotic resistanceVector has been cut in the image to allow fragments to be insertedChromosomal DNa that has been cut with restriction enzyme which give it same ends as the ends of the plasmid, ligates. We are producing a libraryMolecules transformed to yeast cellsMolecles enter the cells, b/c they contain selectable marker, we plate them, every cell that picks up plasmids has complimented whatever is plated onWe also need plasmid to replicate int eh cell that it transformed, so as the cell grows in the colony, every cell in the colony inherits a copy of the chromosomeBack to me:We are producing a library – taken all the DNA from an organism’s genome, cut it up intofragments, cloned into vectors, and the tube where the ligation occurs essentially has a different piece/fragment of the genome. All of this DNA can be grown in E. Coli if necessary. Molecules enter the cell, containing a selective marker. Ex. Plate them with uracil free plate, because they have transfsoremd and recombinance allows them to produce own uracil. Every cell in the colony that grows out from the Dna should replicate thechromosomes and contian cells that can all also produce own uracil. Need a replicator and selective marker. Bascially a piece of DNA that carries info to tell proteins in the cell where replication should happen. Identification of molecules like these are important for idetnfiying reactions that occur. Book: figure explanation – (genetic identification of replicators) a plasmid containing a selectable marker is cut with a restriction enzyme that results in the excision of the plasmid’s normal replicator. So now this fragment has no replicator. Use the same Restrict enzyme on the organism whose replicator you are studying, to isolate the specificreplicator from it, and then ligate that back into the cut plasmid such that you get a new circular plasmid that includes a single fragment derived from your test organism. This DNA is then transformed into the host, and recombinant plasmids are selected using a selectable marker on the plasmid. Cells that exhibit the selectable marker trait indicate that the plasmid can replicate in the cell and must contain a replicator. (basically a test) Isolate the plasmid from the host and sequence the inserted DNA to allow the identification of the sequence of the fragement that contains the replicator. The Eukaryotic Cell Cycle: The major events of cell division,DNA replication and chromosome segregation, are separated in time, into cell cycle phases.During the Gap phases (G1 and G2) checkpoints ensure that all is well with the DNA and segregation machinery, before allowing DNA replication (S phase) and mitosis (M) to commence. The major regulatory decision to enter a new cell cycle occurs in G1.Once S phase has begun, the cell iscommitted to completing the cycle.-In G1, the chromosomes are unwound, andprepared for replication. This is also where celldecides if it wants to go on to start a cell cycle(Depending on factors like availability of nutrients,damage, etc.)Assembly of the Pre-replicative Complex:-The origin recognition complex (ORC) is a complex of 6 proteins that binds to eukaryotic replicators. It recruits Cdc6 and Cdt1, which in turn, recruits and loads the helicase, or MCM complex (Mcm2-7) on DNA in preparation for the S phase – call this entire complex the pre-replicative complex (pre-RC), which is assembled and activated exactly one time in each cell cycle.Book:ORC definition: in eukaryotic ells, the initiator is a six-protein complex called the ORC(origin replication complex). It recognizes a conserved sequence and binds and hydrolyzes ATP(required for sequence specific binding at origin and for ORC to participate in