DNA Repair Mechanisms

DNA repair mechanisms DNA repair Maintaining genetic stability requires not only an accurate mechanism of DNA replication, but also mechanisms for repairing DNA damage These mechanisms are collectively called DNA repair 127 Repair mechanisms Prevention of errors before they happen Direct reversal of damage Excision-repair pathways Postreplication repair 128 129 Reactive oxygen species Some enzymatic systems neutralize potentially damaging compounds before they even react with DNA One example of such a system is the detoxification of reactive oxygen species and oxygen radicals, which are produced during oxidative damage to DNA 130 Superoxide dismutase Catalase 131 8-oxodG The enzyme product of the mutT gene prevents the incorporation of 8oxydeoxyguanosine (8-oxodG) into DNA 8-oxodG is formed from free radical attack of DNA and pairs with A rather than C 132 133 Cyclobutane pyrimidine Some lesions can be repaired by reversal of DNA damage UV light that hits DNA results in the formation of a covalent interaction between two adjacent pyrimidine bases forming structures known as cyclobutane pyrimidine dimers, most frequently between two thymines This product is a mutagenic photodimer Photolyase Cyclobutane pyrimidine can be repaired by a photolyase that has been found in bacteria and lower eukaryotes but not in humans 135 136 Types General excision repair Coupling of transcription and repair Specific excision pathways 137 General excision repair AKA: nucleotide excision repair This system includes the breaking of a phosphodiester bond on either side of the lesion, on the same strand, resulting in the excision of an oligonucleotide In bacteria, the UvrABC protein complex does this work. A helicase removes the strand. The gap is filled by DNA polymerase (in bacteria, it is DNA pol I), and a ligase seals the breaks 138 In human… In human cells, the process is more complex than its bacterial counterpart. However, the basic steps are the same as those in E. coli Defect in this mechanism causes a condition known as Xeroderma pigmentosum 139 Transcription and repair In both eukaryotes and prokaryotes, there is a preferential repair of the transcribed strand of DNA for actively expressed genes RNA polymerase pauses when encountering a lesion The general transcription factor TFIIH and other factors carry out the incision, excision, and repair reactions Then transcription can continue normally 140 141 DNA glycosylase repair pathway DNA glycosylases do not cleave phosphodiester bonds, but instead cleave N-glycosidic (base-sugar) bonds of damaged bases , liberating the altered base and generating an apurinic or an apyrimidinic site, both called AP sites The resulting AP site is then repaired by an AP endonuclease repair pathway 142 DNA glycosylases Numerous DNA glycosylases exist Example: uracil-DNA glycosylase, removes uracil from DNA Uracil residues, which result from the spontaneous deamination of cytosine can lead to a C→T transition if unrepaired. The AP endonucleases cleaves the phosphodiester bonds at AP sites An exonuclease removes the strand, DNA polymerase I fills in the gap, and DNA ligase and re-forms the bond. 143 GO system mutM removes 8-oxodG, or GO, lesion from DNA. If DNA is replicated, mutM first removes the GO lesion Then, mutY removes the mispaired adenine from the opposite strand, leading to restoration of the correct cytosine by repair synthesis (mediated by DNA polymerase I) and allowing subsequent removal of the GO lesion by the mutM product 144 145 Mismatch repair Some repair pathways are capable of recognizing errors even after DNA replication has already occurred One such system, termed the mismatch repair system, can detect mismatches that occur in DNA replication 146 The mechanism Recognize mismatched base pairs. Determine which base in the mismatch is the incorrect one. Excise the incorrect base and carry out repair synthesis This is mediated by the mut protein system. BUT…How can the mismatch repair system determine whether G or T is incorrect? MutS MutL MutH Ssb 147 DNA methylation DNA is methylated following replication by the enzyme, adenine methylase However, it takes the adenine methylase several minutes to methylate the newly synthesized DNA The mismatch repair system in bacteria takes advantage of this delay to repair mismatches in the newly synthesized strand 148 In humans The mismatch repair system has also been characterized in humans. Two of the proteins, hMSH2 and hMLH1, are very similar to their bacterial counterparts, MutS and MutL, respectively. 149 Hereditary nonpolyposis colon cancer (HNPCC) Several genes responsible a type of colon cancer have been identified, including MSH2 and MLH1 The protein products of the MSH2 and MLH1 genes appear to have critical roles in the recognition and repair of DNA mismatches They are homologous to the mutL and mutS genes in bacteria and yeast 150 SOS system A large number of mutagens such as ultraviolet light damage one or more bases, resulting in a replication block, because DNA synthesis will not proceed past a base that cannot specify its complementary partner by hydrogen bonding In bacterial cells, such replication blocks can be bypassed by inserting nonspecific bases. The process requires the activation of a special system, the SOS system 151 The SOS mechanism DNA polymerase III pauses at a type of damage called a TC photodimer This region attracts singlestrand-binding protein (Ssb), as well as the RecA protein, which signals the cell to synthesize the UmuC and UmuD proteins forming the SOS system The SOS system allows DNA polymerization to continue past the dimer 152 SOS system and mutations However, the SOS system inserts the necessary number of bases (often incorrect ones) directly across from the lesion and replication continues without a gap SOS system often generates mutations 153 Recombinational repair The recA gene also takes part in postreplication repair Here the DNA replication system pauses at a UV photodimer or other blocking lesion and then restarts past the block, leaving a single-stranded gap In recombinational repair, this gap is patched by DNA cut from the sister molecule 154 Breast cancer Mutations in BRCA1 account for 2% of all breast cancers and, at most, 5% of ovarian cancer (men are also at risk for breast cancer) BRCA1 activates homologous recombination repair of DNA double-stranded breaks). BRCA1 is also involved in transcription and transcriptioncoupled DNA repair When its function is lost, genetic defects accumulate, which are directly responsible for cancer formation 155 Controversial issue Gene repair Components of the human genome 157 Repetitive DNA sequences 158 Tandem vs. dispersed 159 Satellite (macro-satellite) DNA Repeats of 100 to 6500 bp Tandemly centromeric repeats (171 bp) unique to each chromosome Each chromosome has its unique sequence possible to make DNA probes specific to each Telomeric repeats 160 VNTRs (minisatellite) Mini satellite sequences or VNTRs (variable number of tandem repeats) They are composed of 20 to 100 bp repeats 161 STRs (microsatellites) STRs (short tandem repeats) composed of 2 to 10 bp repeats 162 Polymorphisms of VNTR and STR The number of repeats in micro and mini satellites are highly variable (polymorphic) Useful in: Locating genes in DNA (gene mapping) DNA profiling for paternity testing, forensic testing, confirmation of relatedness and dead body identification 163 PCR of VNTRs VNTR blocks can be extracted and analyzed by RFLP or PCR and size determined by electrophoresis 164 Microsatellites and VNTRs as DNA Markers VNTR in medicine and more The picture below illustrates VNTR allelic length variation among 6 individuals. The likelihood of 2 unrelated individuals having same allelic pattern extremely improbable 166 Real example SINEs and LINEs Two thirds (66.7%) of the repetitive non-coding DNA sequences that are dispersed throughout the genome Divided into short and long interspersed sequences, SINES and LINES. LINES: 7000 bp in length Represent about 4% of our total human genome SINES: Shorter interspersed elements 90 to 500 bp in length Example: Alu sequence (~300 bp) Alu sequences are unique to humans (and some apes) 10% of the total human genome 169 Single nucleotide polymorphism (SNPs) Another source of genetic variation Single-nucleotide substitutions of one base for another Two or more versions of a sequence must each be present in at least one percent of the general population SNPs occur throughout the human genome - about one in every 300 nucleotide base pairs. ~10 million SNPs within the 3-billion-nucleotide human genome 170 Categories of SNPs 171