Biochemistry 401 Lecture 35 Today we re going to talk about replication We re going to talk about replication in bacterial and eukaryotic systems and it s important to keep these two straight We re going to discuss key enzymes and proteins that are involved in replication and also the regulation of this process We re then going to talk about topoisomerase one and two specifically So let s get started Hi Today we re going to talk about replication We re going to talk about replication in two model systems E coli for prokaryotes and humans for eukaryotes The replication is a little bit different in each of these systems so it s important to get this straight So as with any system it s important to think about things in a big picture perspective and for replication we want to concentrate on these things First of all what is happening When does this happen How does it begin How is this beginning regulated What enzymes are doing this Are there any accessory proteins that help these enzymes to know where to start and be able to continue What is the directionality of these enzymes do they go five prime to three prime Three prime to five prime It s important to know this So with this in mind let s get started Replication is DNA synthesis in preparation for cell division Replication is semiconservative This means that the parent DNA separates and a daughter strand is made from each parental template so that the parent cell keeps a parental strand and a new daughter strand and the daughter cell gets a parental strand and a new daughter strand as well The process of replication in bacteria and in humans requires similar things In each replication requires a DNA template So in this case it s the parental strand and it requires ribonucleotides to make RNA primers because first you must make RNA before you can make DNA Deoxyribonucleotides for DNA synthesis are also needed and finally polymerase enzymes accessory enzymes and proteins are also required So those are the things that are similar but what sorts of things are different In bacteria there is generally one circular chromosome In E coli this is true and there are about 4 6 million base pairs on that chromosome Each circular chromosome has one replication start site and the cell division is based on cell size This is the relative surface area to volume ratio Each different bacterium has its own particular optimal size and once that size is surpassed it s time to divide In eukaryotes for instance in humans the chromosomes are linear There are 46 linear chromosomes in humans and approximately 6 billion base pairs That s a lot more DNA that must be copied during replication and as you d expect there are multiple replication start sites to get this big job done quickly Cell division is highly regulated and differs with respect to cell type developmental state intracellular signals and extracellular signals Let s talk about replication in E coli first because this is a little bit easier to understand First of all where does replication start on the chromosome and what sort of enzymes are responsible for this beginning In E coli the initiation of replication occurs at a place called the origin of replication That makes sense doesn t it It s abbreviated ori and in E coli it s oriC The oriC is a DNA sequence in E coli that consists of two main regions The first is an AT rich region The AT repeats represent a site at which the DNA is easy to melt because there are only two hydrogen bonds between AT base pairs and so it s relatively easy to melt The second portion is the DnaA binding site This is a site to which DnaA a binds This DnaA recognizes the sequence and it binds in these specific spots and uses ATP hydrolysis to power the separation of the DNA strands at the ori locus So now that the DNA is separated it s important to make sure that this DNA does not reanneal or form higher order structures like stem loops or things like that We don t want our DNA to get bound up in knots and so we re going to cover this DNA with something called single strand binding protein SSB that coats the DNA and prevents it from reannealing or forming higherorder structures The next thing that happens is DNA a is going to help recruit DnaC and DnaB so it s important to know your ABCs of bacterial replication DnaA binds first DnaB is loaded on to the DNA by DnaC What does this mean DnaB is a helicase that consists of six individual subunits that comprise a ring like structure DnaC opens this up and helps to place this DnaB on the chromosome at the start site of replication So DnaA binds first helps to separate the strands DnaC binds and helps to load DnaB the helicase onto the DNA So DnaB is associated with the primates This primase is called DnaG in E coli and this primase is going to synthesize RNA primers that help get replication going because remember DNA synthesis cannot happen without a primer first and in this case we re going to use an RNA primer RNA first then DNA So DnaG is bound to DnaB and DnaG is the primase and it synthesizes a short strand of RNA as a primer and then DNA synthesis itself can get going As we can see from this illustration the DNA is going to be synthesized in two directions There are actually two primosomes made up of DnaB and DnaG that are found on this chromosome There s one going to the left and one going to the right and so we re going to be replicating in two directions at once Now now the place at which this DNA is separated okay on either side is called a replication fork and it s called a replication fork because it looks like a fork in the road right and so we re going to have one to the left and one to the right and right now we have the primosome bound DnaB and DnaG This is going to help to recruit the main replicative enzyme which is Polymerase III and this enzyme is part of what s called a replisome The replisome itself consists of three main parts we have a beta 2 clamp that helps to hold the polymerase enzyme on the DNA strand and we also have the polymerase itself of course so Pol III core polymerase contains three portions an alpha which is the actual catalytic portion the theta which frankly we really don t know what it does and the epsilon The epsilon actually is the exonuclease capability So if this polymerase makes a mistake the epsilon can take off that nucleotide and give the polymerase another chance to get the right one there but we have to have something that s going to open and close the beta 2 clamp to
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