weber uiuc edu 19 October 2007 MCB150 Lecture 23 Lecture 23 19 October 2007 Announcements More broken hand notes Exam key posted by 2pm Questions are to be brought directly to him Questions about grading the exams are to go to Alejandra Stenger The average for Exam II always dips a bit The first unit material is largely review The average always picks back up for Exam III Mistake Correction We ended Monday s lecture talking about the proofreading ability of DNA polymerases o Proofreading is there to catch mistakes o The 3 5 exonuclease activity of the DNA polymerases are used to correct problems o RNA polymerases do not have the ability to proofread Therefore transcription is inherently more error prone than replication Nor is there proofreading with primase o Also proofreading doesn t always catch all of the mistakes So then what The mismatch repair system The mismatch repair system is a complex of proteins enzymes and molecules The mismatch repair system is constantly scanning DNA looking for mis paired base pairs o E g C T A G etc During replication the new strand is not yet marked We will look at how E coli does it because it is well studied and well understood o Every time there is a 5 GATC 3 eventually a methyl group will be added to the adenine o New DNA adenines gets methylated 10 minutes after replication addition of the adenines o Methylase will recognize the GATC and add the methyl group to the adenine o Brief time between synthesis and methylation This permits distinguishing between old and new and can be used for proofreading The unmarked one is the template while the marked one is parental o Hemi methylated DNA is the partially methylated DNA It looks for the GATC sequences o These are palindromic with their complementary sequence o That is it reads GATC in both directions on the sequences when read from 5 3 In the diagram we see leading and lagging strands near the replication fork with hemimethylated DNA o Some of the regions have not been methylated yet both in the leading strands and in the Okazaki strands o This lets the repair enzymes distinguish which is the correct strand o If it cannot tell them apart the error correction will be wrong 50 of the time Page 1 of 6 weber uiuc edu 19 October 2007 MCB150 Lecture 23 o It always assumes the parental strand is correct The repair enzyme uses the methylation to determine which strand is the template and which is the newly synthesized strand o By default it is assumed the template strand is right o Any changes there will be permanent mutations and those mistakes will be considered right too The repair enzymes look for the unmethylated region and the lag time gives us a window of time to do the repairs Once it is methylated then any repairs will have to guess at the strand o It will still try to fix the error but will guess which is the correct parent strand o Obviously it will get it wrong 50 of the time Mismatch Diagram Do not need to know the MutX names of the repair system But we do need to know what it does DIAGRAM There is a thymine opposite a guanine MutS recognizes the incorrect base pair The MutH will recognize the nearest correct GATC sequence and identify the correct template strand In the diagram the small M indicates the methylated adenine o It won t be far away GATC sequences occur frequently o MutH recognizes which one needs to be fixed o It makes a nick in the strand that is unmethylated Another element will then strip out everything from the GATC through the error o MutL recognizes the strand between the GATC and the problem It is the linker between the problem and the strand to fix o Strip out the erroneous DNA from the GATC to the error After excision DNA pol III will come through again and fill in the hole DNA ligase will seal the nick o It is unlikely that the same mistake will be made twice but as long as it remains unmethylated it can be repaired again Why strip out so many bases perhaps up to a thousand bases o It is more efficient to strip out many of them than it is to do just one DNA pol III can do thousand or so per second It takes less time to do this than to try to replace just one base o Even though this introduces a larger chance of making another mistake the chance is very small Remember though things don t go wrong all that often The chance of replacing 200 bases being repaired correctly is very good How Good is Mismatch Repair 100x more likely to have mutations without the mismatch repair system o The most common type of colon cancer can be traced to a mutation in the human mismatch repair system o It isn t the mismatch repair system that causes the cancer but without it your colon cells may produce mutations that aren t caught that lead to cancer Page 2 of 6 weber uiuc edu 19 October 2007 MCB150 Lecture 23 Matched with proofreading get a 10 000x decrease in error rate o Each type of repair proofreading and mismatch repair by itself reduces errors by a factor of 100x What about changes that happen at some other time not during replication o E g C T at random time due to chemicals or radiation o Anything that damages DNA can result in a mutation that will be passed on Proofreading and mismatch repair only happen at the time of replication o What happens during the rest of S phase Or in G1 G2 o There are other mechanisms that will kick in Multiple layers of protection will attempt to fix the problem as best it can Not every DNA repair mechanism is 100 accurate SOS repair attempt to repair even when really bad knowing that the other choice is to die o Sometimes if there is a major stall in replication or transcription it is due to a mutation and the cell will die if the proteins aren t made in time o Therefore sometimes it is necessary to do a patchwork job to fix the problem to get the cell working again o There is a better chance of surviving and dealing with the introduced problems than to let things go where death is assured The textbook mentions a few others like photoreactivation dimer repair etc The ones mentioned here are the important ones for MCB150 Four General Mutation Categories 1 Base Substitutions substitute one base for another a Missense mutations b Nonsense mutations c Samesense or silent mutations 2 Frame shift 3 Large scale insertions 4 Large scale deletions Mutation Categories 1 2 are point mutations o SMALL changes of one or two bases 3 4 are large scale and will contain large chunks of rearranged DNA o Insertion and deletions involve LARGE chunks of DNA