in the last part of lecture seven I wanted to tell you about DNA repair mechanisms here I've summarized some of the different repair mechanisms first mismatch repair this catches errors in base pairing the wrong base pairs being formed in DNA that escape proofreading by DNA polymerase um so it's going to scan along the chromosome and look for the wrong base pairs and it needs to distinguish which strand of DNA was just synthesized so it can do that looking at the methylation levels on the DNA this is something that occurs in both procaryotes and ukar methyl groups are added to certain bases and this happens after replication so the newly ize strand will have much fewer methyl groups that have been added than the parental strand it would be assumed the parental strand is the correct sequence so the newly made strand can be fixed mismatch repair will utilize enzymes to detect the mismatch other enzymes to actually cause a single strand break in the DNA Nick the DNA remove the base and replace it with DNA plase and DNA ligase in the next slide we'll look at a diagram going through the process of mismatch repair with these various enzymes post-replication repair this fixes lesions on the DNA and areas not replicated using recombination uh SOS repair this is the last resort means a fixing mismatches and gaps DNA synthesis is very error prone here we're not going to talk too much about SOS repair go ahead and read about that in your textbook in chapter 16 starting with mismatch repair here on the DNA there happens to be a mismatch sometimes errors escape proof reading of DNA polymerize 3 and it's the job of this repair system to scan the chromosome after replication and find these mismatches and fix it this is a figure from another textbook different proteins are needed to complete mismatch repair here we have three different proteins that have come to this area of mismatch first the mismatch needs to be detected in a coli that is the job of a protein called mute s mute s then recruits two other proteins mute L and mute H mute H is an endonuclease and what it's going to do is it's going to cleave the DNA nearby not exactly at but near the mismatch so here it just breaks the backbone breaking a phosphodiester bond and then another enzyme exonuclease one here in step four will come in and it will remove these bases um so it'll go to the break and the DNA and it just starts removing one base at a time it'll remove a section including the highlighted uh base here T removing that mismatch okay so these enzymes know which strand of the DNA to break because here the blue strand is identified as the new the newer strand having less methylation after the section of the DNA is removed our typical DNA pmer c will come in it will resynthesize and the Gap will be sealed by DNA ligase so a couple different enzymes are mentioned here we have an endonuclease and exonuclease let me just distinguish between these two really quick an endonuclease cleaves the backbone of one strand of DNA so literally an endonuclease will break a phosphodiester bond the other enzyme an XO nucleas um this is going to act at the end of a DNA strand or add a break and what the exonuclease is going to do is it's going to remove one base at a time it can remove many bases okay so we have an endonuclease cleaving the backbone a exonuclease acting at that cleavage site to remove bases one at a time so M mismatch repair mute s mute L mute H detect the mismatch cleave the backbone remove part of that newer DNA strand and give the cell a chance to resynthesize it hopefully making the correct uh base pairing following the base pairing rules okay so this can take care of those low levels of random mutations that occur during replication well how about some of the other damage caused by chemical mutagens that actually make chemical changes to the bases basic exision repair this can uh find and remove modified bases uh eight oxoguanine this would be the result of oxidation of a guanine three methyl adenine uh would be the result of alkal of an adenine UIL remember if there's a uracil in DNA that occurs from deamination of ayto so these are all modified bases a result of some of those simple chemical modifications we talked about previously so again we have a figure from another textbook here highlighted in blue is the modified base in eoli this modified base would be detected and removed by an enzyme called Al a in general that base is removed by a specific glycosylase and a glycosylase is going to cleave the modified base from the sugar so it simply removes the base so here we have the backbone of the DNA the Bas in blue DNA glycos removes the base the backbone is still intact so this bond that connects the sugar to the base remember that's the glycosidic bond so a DNA glycosilated is going to break the glycosidic bond holding the base to um the sugar or DNA backbone okay so when that happens this creates an AP site AP P standing for puring or perimidine we have an apurinic or an aidic site a meaning without because that's what just happened we removed the base um basic cision repair can also detect spontaneous AP sites where a base just happened to be lost if a glycos bond spontaneously um broke so next here um we need an endonuclease to come in remember an endonuclease is going to break the backbone because here in the backbone we have the remnants of that nucleotide we remove the base but there's still the sugar and phosphate in the backbone that has to be removed so it can be replaced AP endonuclease breaks the backbone um and so that actually will cut out the the sugar and phosphate and now that lost base can be replaced with DNA polymerase so the excision repair system would be activated DNA polymerase will come in step five replace the one base and this final phosphodiester Bond must be formed and that's the job of DNA ligase number six okay now a specific example of basic cision repair involves the repair of uracil and DNA remember ell should be in RNA not in DNA ell and DNA is a result of deaminated cytosines and these are detected and fixed with basic cision repair utilizing the specific enzyme uracil DNA glycos so here we have a GU based pair it should be a GC based pair but this U is a deaminated c this is detected by your cell DNA uh glycosidase and it would remove The UU because it breaks a glycosidic bond removing the base then the basic exision repair system um that can be abbreviated beer um an AP endonuclease comes in and it's going to cleave the backbone to get rid of the sugar and phosphate there the backbone is cleaved and now DNA plase 1 can replace the base DNA ligase can seal it to the other side of the DNA strand so this prevents a GC based pair from becoming an at based pair or mutation now other damage to the DNA may be more bulky than just some of these examples listed here if you have oxidation a small alkal group such as a methyl group getting added or if you have um if you lost an amino group FR if you have um deamination okay so for more bulky damage another system the nucleotide excision repair is going to come in so this would be bulky lesions or modified bases um not otherwise repaired okay so any modified bases that were not repaired by the base excision repair system can be targeted by nucleotide exision repair otherwise abbreviated Neer so in nucleotide exision repair we have a lesioned area and what happens when we think about excision the title of this repair excision literally means to cut out and that's what happens in this repair mechanism um there is a nuclease this is an enzyme that will cut out a section of one strand of DNA so the nucleas excises or cuts out the lesion on that one strand DNA plase 1 will remake the strand DNA ligase will seal this final phosphodiester Bond uh this can be very important this uh lesion an example of that lesion could be a thyine dier okay it could be other modified bases could be a thyine dier this is found in humans and also in bacteria in humans this system the nucleotide excision repair system requires 30 genes to be functional so this forms a large protein complex to uh form the nucleas to excise Aion and then utilizes enzymes of DNA replication to replace the removed DNA strand some genetic disorders result if any of these genes are mutated so the fun so the protein is nonfunctional here are two examples zerod Derma p mosum XP these individuals lack UV repair exposure to sunlight results in mutations here we have an individual who's affected um they would have lots and lots of melanomas they have a high incidence of skin cancer they need to avoid the Sun as much as possible because they cannot repair uh UV damage or exposure to nonionizing radiation another disorder caused by mutation in a different Gene within the system is cocaine syndrome these individuals are also photosensitive although they see less tumor formation um they're characterized by neurological and growth Disorder so here is a picture of a boy who has cocaine syndrome so in this picture that I found online we have the caption a effect in the ability of cells to repair DNA that is being transcribed so these repair systems are very important if they are mutated they can lead to problems such as cancer or other growth disorders so there are other ways that thyine diers can be repaired remember thyine diers occur as a result of UV exposure in bacteria or procaryotes um they can use photoreactivation repair shown in this diagram here is the DNA if it is acted on by UV light so that is our DNA damage so UV light is going to hit the DNA and forms these calent bonds causing a thyine dimer this is a lesion in photoreactivation this is also called light repair this utilizes an enzyme only found in bacteria called photol lias and that enzyme uh it utilizes energy in light to make the most simple repair of the stying dier it simply breaks those calent bonds restoring it to to its normal DNA structure okay now note this enzyme is not found in humans this is a repair mechanism light repair or photo reactivation found in bacteria or Pro carots so in humans they could use nucleotide excision repair now what if this damaged area If This Were A thyine dier what if that's not repaired well this stretch of DNA cannot undergo transcription because this is a block to being copied by RNA plase nor can it undergo DNA replication this will also block copying by DNA polymerase needed for DNA replication okay this brings us to our next repair mechanism post-replication repair and this utilizes recombination here on the DNA here is DNA ready for replication we have a thyine Diemer or alion this is not repaired so this thyine dimer cannot be used by DNA polymerase to synthesize the complimentary strand so if we follow the numbers here here we have the replication fork formed for replication this bottom strand is unaffected it'll be replicated normally we can see here with the arrows the bottom strand is the leading strand the top strand however the lagging strand when DNA polymerase gets to this area of damage this thyine dimer it will just skip over it so there's a number of there's a section that's not being synthesized within that DNA strand so there's a gap post replication repair its job is to fix this Gap and what it can do is it can utilize Rec combination with the other replicated strand so that it has a template for synthesizing these missing bases on the purple strand so the pink strand containing the thy dimer cannot be used as a template because of that thyine dier however recombination can utilize um the newly made strand um here in purple on the bottom as uh the template so here we have recombination it actually remember recombination is where uh sections of DNA are exchanged so um we have DNA from the other strand being exchanged and now that Gap that was formed in the other strand is resynthesized so the so you have a good template here to fix that Gap post-replication repair means that it's after replication DNA pyas has already passed but this Gap can be fixed utilizing recombination so recomb happened here where U the area highlighted in green was transferred to fill the Gap and that new Gap that's formed can simply be filled in by DNA plase and sealed by DNA ligase okay so recombination can be used to fix some DNA damage this can also be used if there are breaks in the DNA the last figure that I want to show you here deals with double stranded DNA breaks so dsdna refers to double stranded DNA double stranded DNA breaks these can be caused by what exposure exposure to ionizing radiation um so uh exposure to gamma w so ionizing radiation this would be radioactivity here we have a double stranded DNA break it may not be a clean break you can see each strand is broken somewhere slightly different um so double stranded DNA breaks in this case we're looking at um using Rec combination with a sister chromatid okay now sister chromatids these exist after replication so this diagram is showing repair that occurs after replication but before cell division okay so if this were bacteria you would have the two chromosomes so you would have one copy of its circular chromosome and then you would have the second copy of its circular chromosome If This Were human cells then the duplicated chromosomes appear um like an X so in humans uh these would be sister chromatids you would have one chromosome and it's copied chromosome and these would look like this during metaphase okay so sister chromatids would be these two copies of the same chromosome so you can have recombination between copies of the chromosome or cister chromatids so what happens is if here they've labeled all the inss so if we look at this second portion here and add to that remember DNA if one end's three prime the other end has to be the five Prime end now why is that important that's important because DNA synthesis occurs by adding nucleotides only to a three prime end so we have this part that can be extended and we have on the top strand a three prime nend can be extended uh recombination is used the thre Prime n inserts into the copied chromosome or sister chromatid and it uses the sister chromatid as a template to extend or synthesize off that three prime end so here DNA is added this then rejoins its chromosome and so now this provides a template for synthesis of the top strand okay so looking at this diagram we have in the middle here we have recombination occurring but not in the typical sense where DNA is being exchanged in this case as the DNA in and the pink DNA invades the blue DNA molecule it's using the blue DNA molecule as a template for DNA synthesis so um here we have DNA synthesis which is already highlighted for you um then the heteroduplex is resolved recombination ends and now that bottom strand which is complete is used as a template for synthesis of the top strand so these gapped portions are filled utilizing um recombination so in this case where combination doesn't go all the way through to exchange the blue with the pink chromosome instead you just have strained Invasion and that assists for DNA synthesis by providing the template for synthesis okay so you can see with chapter 16 it's very important that you understand DNA structure in some of our previous lectures so that you can understand how the DNA can be altered resulting in mutations and also how it can be corrected as you read through this chapter chapter 16 take a look at questions 2 through 16 go through those uh if you have questions work with your group members or maybe post on discussion board to get help from your classmates