in the second part of lecture seven I want to talk to you about how specifically DNA damage occurs we're going to look at some of the changes that can occur to the DNA resulting in point mutations frame shift mutations or other types of DNA damage sometimes errors and replication occur where DNA polymerase inserts the wrong base and doesn't follow the base pairing rules you might be wondering how this can be possible remember the bases interact by hydrogen bonds here we have the standard base pairing following the base pairing rules and the dotted lines show hydrogen bonds between the bases so here we have a thymine base pairing with an adenine what's highlighted here in red are the functional groups the chemical groups that participate in hydrogen bonds so inamine the keto group is important this is a carbon double bonded with an oxygen and in adenine the amino group is important this nitrogen uh being bound to hydrogen so nh2 here is an amino group cble Bond O is a ketone group so we have a hydrogen bond between the oxygen and the hydrogen here similar Sly in base pairing between cytosine and guanine there are hydrogen bonds being formed between um here we have a keto group at the top I'm sorry an amino group keto group at the bottom um on guanine we have a ketone group and then an amino group at the bottom also highlighted within the Rings themselves are some other areas some these have double bonds these two lines indicate two electrons are being shared forming a double bond with double bonds those um shared electrons can undergo resonance and that's what we'll see in Part B where you have Resonance of the double bond so if we first focus on thiamine on this keto group double bond to an oxygen that can have resonance um so this double bond can sometimes be form between carbon and nitrogen instead of carbon and oxygen and that's what we see in Part B the double bond has shifted and now we have thymine in the enol form so instead of carbon double bond to an oxygen this picked up an extra proton and now we have an O here and that becomes um different hydrogen bonding partner so instead of having a oxygen here to participate in a hydrogen Bond we have a hydrogen so this is thiamine in the Eno form and very simply the chemical reason is you can have resonant structures around double bonds and this changes the hydrogen bond Partners so we have different hydrogen bond pattern here now three hydrogen bonds are being formed allowing thyine to base pair it with guanine so this would be thyine in the enol form which is not the normal form form the normal form is the keto form same thing can happen with cytosine cytosine normally in the amino form where you have an amino group here on top there is a double bond within the ring that double bond can have resonance so that it moves to the carbon and nitrogen outside the ring as shown in Part B and when that happens this nitrogen loses a proton here it has only one proton bound instead of two hydrogens bound at the top here and now this nitrogen this can participate in a double bond instead of the hydrogen participating in a double bond so we have different hydrogen bonding patterns according to if you have the normal keto and amino forms versus the anomalous enol and amino forms okay we can summarize this to say simply that the bases themselves can undergo slight change due to Resonance and this changes the hydrogen bonding properties a simple theme that you'll find in this part of the lecture is anything that changes the hydrogen bonding properties of a base will result in different base pairing where the normal base pairing rules are not followed this results in a point mutation if we have a t PA with a G or a c paired with an a that is the wrong base pair one base is changed during replication resulting in a point mutation here's an overview of that here we have DNA undergoing replication each Str is separated we have the normal forms of the bases on the left here we have um a toomic shift to the amino form so this is a anomalous or different form and that will not follow the normal base pairing rules semiconservative replication each strand is copied no mutation with the normal form of the bases with the anomalous form of the base now this a is paired with C this is our point mutation highlighted here in red when this under goes replication again if this is not caught by the cell we'll talk about mismatch repair where the cell does has extra repair mechanisms to detect mismatches but if that's not repaired as this is replicated again for cell division then um this left strand will have a mutation and here we have a transition mutation because what should have been a c uh I'm sorry what should have been a t became a c remember transitions are when um you have uh in this case a perimidine or a pine being replaced by I'm sorry a peridine being replaced by a perimidine so T and C are both Pines remember our peridine memory tool Pines are cut in half they're the smaller bases including c u and T so when a t is replaced with a c that is a transition mutation not a transversion mutation so here we have a mutation as this one strand is copied the other strand if it the a shifted back to the normal form it would form the usual base pair so errors and base pairing lead two point mutation and this can be as simple as some chemical shifts within the base as we saw with those different forms and the Bas toomer this can also occur due to chemical damage to DNA bases here I've summarized different types of chemical damage and we'll look at some figures showing these chemical uh changes that can occur to the bases first is deamination this removes an amino group so an amino group is nh2 those are found normally on the basis and when that's removed say on a cytosine or adenine is replaced with a keto Group which is a carbon double bonded to an oxygen so an amino group would be a carbon with a single bond to a nitrogen or nh2 a keto group is a carbon double bonded to an oxygen oxidative damage this occurs when reactive oxygen species or free radicals attack bases free radicals that are present inside of cells include super oxide O2 minus that minus indicates extra electrons that are very reactive if it contacts the DNA it will undergo a chemical reaction changing the base also included our hydrogen uh peroxide H2O2 another example is the hydroxy radical oh minus all those are reactive oxygen species that can cause oxidative damage third I'd like to tell you about alkala agents these add alkalol groups to either Amino or keto groups on DNA alkal groups these are long chains of carbons and hydrogens um the simplest alkal group can be ch2 okay um they can also be um longer um so any chains of ch2s um so we could have one ch3 we could have two c2h5 if there's just carbons and hydrogens being added that's referred to as an alkal group okay other types of damage can occur um during replication if a normal base is not used but if it's substituted with a base analog that will have different base pairing leading to uh point mutations so here's a little definition for a base analog these are compounds that resemble bases and are used in place of the normal bases purines or permiting during uh nucle I biosynthesis um they have a higher rate of toomic shift leading to wrong base pairs being formed let's go through each one of these starting with deamination here we're looking at deamination of cytosine in part A and what's highlighted in red is the group that's going to change so here we have an amino group deamination is removal of nh2 so if that gets removed it's replaced with this keto group a double bond to an oxygen having different uh base pairing interaction than the amino group okay so the cytosine actually um becomes ell uril base pairs with adenine adenine also have an amino group it can lose that with deamination so here it has lost that we have a keto group instead and now it base pairs with cytosine okay so these are the wrong base pairs so if we think about what kind of mutation would occur normally we would have a CG base pair after deamination we have our modified C base pairing with a okay so a g was replaced with an A and that would be a transition mutation if we look at Adine Adine normally forms an a base pair after deamination this modified a hypoxanthine base pairs with C C and T those are both peridin so again we have a transition mutation now your Cil we know is found in RNA and not in DNA um and there are repair mechanisms that we'll talk about in the next part of this lecture that will fix ills found in DNA and a question that sometimes you may think of is well why is thiamine Us in DNA and not urasil here we have thiamine and the uel base and you can see the only difference between thine and uel is this extra ch3 methyl group on thiamine it's a little tag to identify thyine is different from uracil now I've also included cytosine here okay looking at thyine if we look at the groups on this ring we have um two Ketone groups this double bond to an oxygen and we have this methyl group osil simply has the two Ketone groups and cyto scine if we look at that it has an amino group and a ketone group now cytosines in the DNA can undergo spontaneous deamination where this amino group uh is lost and it's replaced with a double bond to an oxygen or a ketone group so if that happens cytosine when it undergoes deamination it becomes uril so any you or your cell in the DNA the cell knows should be a cytosine and this is because uh any uracils they lack this methyl group ch3 and so on thiamine that methyl group is used to distinguish thines from deaminated cytosines so this is one simple difference in DNA that helps preserve the Fidelity of DNA helps to preserve the information and minimize mutations okay so d D aminated cytosines your any uracils in the DNA those are fixed with base excision repair which we'll talk about in the next section uh utilizing a special enzyme that recognizes uril and DNA uril DNA glycosylase another type of point mutation can be um induced with the action of Base analoges in this figure we're looking at the normal base thyine and we're looking at two different or one analog in two forms so fibrom osil is an analog and a base analog is a chemical that closely mimics a normal base so we can think of this as an analog or a mimic these are chemicals that if they are present when nucleotides are being formed these chemicals fibrom muril may be used in place of thyine to form a nucleotide now this is problematic because fibrom muril has this bromine atom not found normally on thyine and based analoges tend to go back and forth between their different toomic forms keto and Eno forms at a higher rate or higher frequency than the normal base so the base analog in one form can base pair with adenine which would be uh the normal base pair but in its Eno form it base pairs with guanine so this would be um causing a mutation so thine normally participates in a TA base pair in the mutation at the bottom when we have five bromoil now base pairing with guanine this is a mutation we've gone from having an a as a base pairing partner to a g as a base pairing partner both A and G are purines so this would be uh a transition mutation other chemicals act as alkala agents in this figure we're looking at the action of the chemical EMS another example uh could be mustard gas um that's an example of an older chemical warfare agent um used to basically kill people um and an alkala agent is going to add alkal groups here highlighted in red is the modification an alkal group is any chain of carbons and hydrogens so here we have c2h5 and this gets added to um this oxygen or this keto group okay so guanine normally would form a GC base pair now we have the alkal guanine it forms six ethyl guanine eth refering referring to ch2 H5 so I'll just put that g in quotes and it now pairs with T so a GT base pair we have substituted a c with a t but both C and T are peridin this is a transition mutation so these three examples that we've talked about alkala agents based analoges and deamination are all specific examples of damage to the DNA that causes point mutations there's other types of DNA damage that can occur transposants these are also known as jumping genes these are sequences of DNA that can copy themselves and insert different spots in the genome if they insert within a gene they can destroy the actual protein that is encoded by that Gene this can lead to a null mutation where the cell can no longer make whatever protein was encoded by that Gene where the transposon inserted itself in the genome intercalating agents these are other chemicals these insert in the DNA Helix between bases and they cause the Helix to bulge many times intercalating agents result in a frame shift mutation occurring during either replication or repair of the DNA remember a frame shift this occurs when bases are accidentally deleted or added UV light this is considered nonionizing radiation this makes changes to ad adjacent peridin remember the peridin are the C's and the t's especially thines and this can cause thiamine diers also known as perimine diers now we're going to look at most of these in a little bit more detail last we have ionizing radiation as opposed to UV light ionizing radiation or exposure to radioactivity this produces reactive high energy particles that actually break the DNA backbone they break phosphodiester bonds causing double stranded DNA breaks or single stranded DNA breaks so we have these two terms nonionizing radiation referring to UV light ionizing radiation referring to exposure to radioactivity um we're not really going to talk about transposons at this moment but let's look at these last three starting with intercalating agents so intercalating agents many times result in frame shift mutations these are chemicals that insert between bases of the double helix here we're looking at a couple diagrams the first in blue we have the backbone of double stranded DNA so we have the two strands of DNA and green are the basis hydrogen bonding here we have an intercol agent this is a chemical that wedges itself between neighboring base pairs if we were to look at a normal double helix the base pairs as you go down the Helix they stack very closely to each other if you add an intercalating agent shown in red that squeezes in between those base pairs that line up one on top of the other and it distorts the Helix so here we can see some Distortion to the Helix so that when this DNA is repaired or under goes replication errors occur bases get skipped or extra bases are added some examples are U some dyes some dyes used in the laboratory to visualize DNA such as aium bromide they make the DNA molecule fluorescent but if you get this on your skin it could you know result in mutations as that cell divides apoxin this is another chemical this is actually made by a mold and ingestion of high levels of apoxin in things like grain are linked to liver cancer so apoxin Works in this way as an intercol agent now what about ionizing and nonionizing radiation here we're looking at the Spectrum of different wavelengths of light in the very center here we have the narrow spectrum of visible light all of our colors then we have UV light x-rays gamma rays Etc so along this spectrum of light there is the nonionizing radiation and these are fairly small it's mostly UV light as we get even smaller these Rays become more damaging so you can see with decreased wavelength smaller waves there's increased energy so when something's actually radioactive it has more energy so looking at gamma rays even smaller these are considered ionizing radiation so each of these are going to cause different types of damage UV light considered non-ionizing radiation versus gamma rays exposure to radioactive substances um forming ionizing radiation diing diers uh are the main result of non-ionizing radiation or exposure to U light so excess exposure to sunlight can provide high doses of UV here we're looking at one strand of DNA and we have two adjacent peridin this is especially common when you have adjacent thyine bases and the UV like causes a reaction between these two bases forming these extra calent bonds so this is a thyine dier caused by UV light nonionizing radiation we just have these two extra calent bonds being formed this is not normal to have coent bonds linking the bases now along the DNA where this occurs in thyine dier this is going to block BL use of this section of the DNA for either replication or transcription uh transcription being needed for gene expression so this is a damage a sort of block put in the DNA that has to be detected and repaired now what about ionizing radiation or gamma rays this is a interesting pie chart taken from another genetics textbook and this shows annual human exposure to radiation in the US so nuclear power plants have radioactivity um different things you can have diagnostic x-rays providing low amounts of radioactivity cosmic rays that was also on the energy Spectrum natural radio isotopes in the soil and by far the most exposure comes from radon gas and radon gas is produced as certain elements in the soil decay um so different areas of the country depending on the composition of the soil will have higher or lower levels of these elements resulting in higher or low lower levels of radon gas being produced now when you think about exposure to radioactivity you might think about something extreme like Chernobyl where there was a meltdown in a nuclear power plant at the end of chapter 16 in the genetics technology ology and Society section the results of Chernobyl are discussed U this happened in the mid 80s in Ukraine and Russia um there were only 62 direct deaths as a result of this meltdown many of those were workers at the um nuclear power plant or some of the First Responders to the crisis they experienced radiation sickness or thyroid cancer resulting in death they ended up closing off about 17 miles all around the plant and uh they claimed that you know there was not huge increases in cancer in those who lived in the surrounding areas more recently uh we have uh the problem that happened in Japan after the earthquake with radiation exposure you might also think about atomic bombs or nuclear weapons um so we know from World War II the bombs dropped on Hiroshima and Nagasaki resulted in increased number of cancers and people who were in the vicinity of those bombs now certain cancers will come about in different times a little later on the class we'll have a whole lecture just on cancer and one of the main High Hallmarks of cancer is that cancer develops typically after a cell has undergone many mutations scientists think as many as 12 mutations or I'm sorry as few as 12 mutations may be needed to convert a normal cell to a cancer cell and then it takes time for that to grow and form a tumor and spread so after uh the atomic bombs used in World War II those individuals exposed within about 2 years there was an increased rate of leukemias these would be uh Cancers and the blood about 10 years later there were an increased rate of solid tumors and as long as 40 years later increased incidence of brain cancer so when we think about mutations or damage to the DNA I've talked to you about two main types of damage those caused by chemicals um so you can have deamination alkal of the DNA oxidation you can have intercalating agents and then we've also talked about radiation nonionizing and ionizing radiation okay now going back to that first part chemicals uh how do they know that chemicals uh certain chemicals cause mutations well one test that is very simple is a good screening test or initial diagnostic test to identify the potential of a chemical to cause mutations and this is CA called the as test the as test was developed by Bruce Ames at UC Berkeley in the 1970s um Bruce Ames is still doing active research at UC Berkeley if you're curious you can look him up um but he's famous for developing this test so this is a test used to see if chemicals may be mutagens or able to cause mutations and this was the first test developed to use a bacteria it uses a bacteria a certain strain of salmonella now prior to the as test if scientists wanted to know if a chemical caus mutations they would have to expose various lab animals to the chemical and wait to see the effects animals have varying lifespans if we think about a mouse it's normal life span is about 2 years so those could be very long tests bacteria on the other hand divide very rapidly your typical bacteria will divide every 20 or 40 minutes so you can see the effects of a mutation in The Offspring of an organism so for bacteria that can be the next day very quickly so the ases test utilizes a very specific strain of salmonella this is a salmonella his minus oxit his referring to the amino acid histadine okay so in the his minus oxit minus it's lost something this bacteria has a point mutation in a gene needed for histadine synthesis so it can no longer make this amino acid it can only grow in enriched media that is supplemented with histadine it cannot grow in minimal media so this was um the test subject the salmonella his minus bacteria how is this test set up the bacteria is exposed to chemicals and the chemical may be mixed with liver extract and the purpose of that is to mimic the normal metabolic breakdown of chemicals in the body um the liver is really the Hub of your metabolism for your body it contains many many enzymes to degrade various drugs and chemicals um that may be ingested so sometimes chemicals themselves are not mutagens but after they're broken down or metabolized in the body by the liver they can become mutagens so mixing the chemical with a liver extract will mimic other chemicals that are produced in the body after the chemical is ingested so what is a positive result a positive result would be the ability of this salmonella his minus to grow on minimal media and that would mean there was a specific mutation a reversion of his minus to his plus so if we go back to the definition of this salmonella his minus oxit an oxit trro just refers to uh nutritional mutant in this case we have salmonella unable to make the amino acid histadine this is due to one base change so if other mutations occur randomly they may change that same base that caus the salmonella to be his minus and they may revert that back to its normal base sequence so a reversion mut is just a second mutation in this case it's a second mutation that converts his minus back to his plus so the his minus would be the mutant form the normal or wild type form would be his plus and a reversion this is just a mutation that converts a mutant back to a wild type wild type being the normal form okay so normally his minus cannot grow on minimal media if it's exposed to chemicals chemicals cause mutations randomly by chance some of those can cause a reversion and if there are lots of colonies on minimal media then that would indicate that lots of mutations occurred from that chemical let me show you a diagram here is the figure from your book with the as tests we have the his minus oxit bacteria Plus liver enzymes um here is the bacteria plus the mutagen or the chemical plus liver enzymes so with any test you need to have a control and your test sample so this first one is the control where you would expect um few mutations and the second tube is the test sample and the question is does the chemical cause mutations okay now in the test sample um the chemical is added to a paper disc and this is placed on a petri dish that has been covered with the salmonella his minus bacteria so in the control there is no disc because there's no chemical it's simply covered with the his minus bacteria and those bacteria have already been mixed with the liver enzymes so here we have a broth of bacteria and that we contain billions and billions of cells so just by random chance if mutations occur you know with each cell there's an additional opportunity for that mutation to affect potentially this Gene for making the amino acid histadine so the control will show just spontaneous mutations each uh growth here each colony would represent a spontan anous mutation so very few if the chemical on this disc were a mutagen causing mutations then you would see lots of growth around that disc so notice the growth is around the disc and not evenly throughout the plate and this indicates that the chemical is causing mutations