MCB 250 1st Edition Lecture 10Outline of Current Lecture 1. DNA2. Overall functions of topoisomerases3. E. Coli Chromosome4. Interaction5. Regulation6. Eukaryotes supercoiled7. DNA replicationCurrent Lecture1. DNA- Movement through a gel is dependent on charge and shape- DNA has different topological states and have different shapes and move through the geldifferently- TOPO 1 type one topoisomerase, break one strand and let the molecule rotate around 1 time and put the strand back together. Maintain energy of the phosphodiester bond by adding tyrosine in hydroxyl group- Doesn’t break and let it go, the molecule would relax. Rather, it grabs a hold of it and allows it to relax by 1. Grab it again and allows it to relax again with a different topo.- Molecules decreasing in linking number and migrate differently in gel. Each band represents the same DNA molecule but in a different topological state (different linking number)- Gyrase changes linking number by -. Everything else relaxes DNA2. Overall functions of topoisomerases:- Control supercoil density. All will relax DNA except gyrase which will introduce negative supercoil- Untangle knotsThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Resolve catenanes: when two circular pieces of DNA, can end up with the two moleculeslinked together. They’re stuck together and you need to separate the two molecules for daughter cells. Topoisomerase type 2 will break one of the molecules pass it through andrepolarize it to two circles. - All cells require topoisomerases. 3. E. Coli Chromosome- Higher order structure in the over chromosome. The massive stuff “nucleoid” and loops of chromosome come out of the region. Don’t know what makes up the nucleoid in the middle. It includes topoisomerase though, which control structure of DNA- The structure doesn’t seem to be statico A given gene in the E. Coli chromosome is not always found anywhere. It is a fluidstructure. Every part of the E. Coli chromosome can talk to any other part. - Less known in E. Coli than higher eukaryote cell- DNA is wrapped around histones. - Nucleosome: DNA wrapped around histones- Histones are small proteins and very basic- Wrap negatively charged phosphodiester backbones with positive proteins- “Core Histones” disk that DNA wraps around are made of 2 copies each. 4. Interaction- Interaction within phosphate backbone and around the minor groove. The histones are homologous to one another due to the folding. Until the DNA is interacting with histonesin a repetitive fashion. The DNA is interacting primarily with phosphates in the backbone. There are some interactions in bases within minor groove. - What would be indicative of protein interacted with DNA with phosphates and a little in the minor groove: NOT sequence specific. Can wrap any piece of DNA around the histones. The interaction with the histones phosphate backbone, some minor groove interactions (do not distinct between base pairs because not a lot of information) Non-sequence specific, but very tight interactions with DNA. Bending the DNA around the histone proteins.- 20-30% of the total mass of the histones is actually hanging out in the debris- Beyond 10nm fiber the DNA is inaccessible to transcription/replication/regulatory. The Level of compaction of chromosome affects the ability of the DNA to be accessible and utilized. Regulated phenomenon- Light microscope allows us to see DNA in 2 states: compact (heterochromatin), loosely packed (euchromatin)o Only euchromatin is expressed. Heterochromatin is not being expressed and is shut down. This is highly regulated and a way to turn genes on and off.5. Regulation:- Can control overall accessibility of DNA- Can imagine that a particular promoter (recognized sequence) in the middle wrapped around histone, inaccessible in 10nm. Go to unpack histone and move the histone over to get the DNA open wherever you want. Proteins and modifications of the histone tails control this.- Very specific phenomenon occurs6. Eukaryotes Supercoiled?- Eukaryotes DO NOT HAVE GYRASE- DNA is not a closed circle, it is linear- DNA is probably attached to nuclear matrix- Wrapping DNA around histones is EQUIVALENT to negative supercoiling- Eukaryotes have both type 1 and 2 topoisomerase to control - Need negative supercoil to unwind to access- Wrap DNA not covalently closed around the histone7. DNA replication:- E. coli chromosome is a single covalently closed circle- There is a single origin of replication: unwind DNA at the ori and build two machines: replication forks. Each will travel in different directions around the chromosome and then run into each other. This duplicates the chromosome into 2. - Count on topoisomerases to sort out all the knots in the end and sort out to 2 daughter chromosomes that can separate. 40 minutes with 4.6 million base pairs- All DNA polymerases require primer and a template!!!- ALL replication proceeds 5’-3’- Use the information from template strand to synthesis the other strand. The other strand is the “primer”. The primer needs a free 3’-OH group. All DNA polymerases will extend the free 3’-OH group and synthesize DNA from 5’ to 3’ using the template strand as the template. - Chemistry: substrates for DNA replication are the 2’deoxytrinucleotides (dNTP) AGCT as the base. But it is a TRI phosphate. Use the energy inherent in the two phosphoanhidride bonds to carry out the reaction. The 3’ OH group of the growing chainis going to attack the phosphate bond between the alpha creating the phosphodiester linkage of the DNA. The other product is pyrophosphate, which is 2 phosphate groups connected together. It is recognized by pyrophosphatase, which will hydrolyze the