8-oxodG • The enzyme product of the mutT gene prevents the incorporation of 8- oxydeoxyguanosine (8- oxodG) into DNA • 8-oxodG is formed from free radical attack of DNA and pairs with A rather than C 52
EXCISION-REPAIR PATHWAYS 53
Types • General excision repair • Coupling of transcription and repair • Specific excision pathways 54
General excision repair • Also termed 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 • This excision leaves a gap that is filled by repair synthesis, and a ligase seals the breaks 55
The mechanism • In E. coli, the UvrA protein recognizes the damaged DNA, forms a complex with UvrB and leads the UvrB subunit to the damage site • The UvrC protein then binds to UvrB • Each of these subunits makes an incision (a cut) • The short DNA is unwound and released by a helicase • DNA polymerase I then fills in the gap, followed by the action of DNA ligase 56
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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 58
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 59
SPECIFIC EXCISION PATHWAYS 60
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 61
DNA glycosylases • Numerous DNA glycosylases exist • One, 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 62
AP endonuclease repair pathway • The AP endonucleases introduce chain breaks by cleaving the phosphodiester bonds at AP sites • This bond cleavage initiates an excision-repair process mediated by an exonuclease, DNA polymerase I, and DNA ligase 63
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 64
POSTREPLICATION REPAIR 65
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 66
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 67
The mechanism in details • MutS binds to mispair. MutS • MutH and MutL are recruited to form a complex • MutH cuts the newly synthesized MutL (unmethylated) strand, and exonuclease degradation goes past the point of the MutH mismatch, leaving a patch • Single-strand-binding protein (Ssb) protects the single-stranded region across from the missing patch Ssb • Repair synthesis and ligation fill in the gap 68
But…. • In a G-T mismatch, both are normal bases in DNA • How can the mismatch repair system determine whether G or T is incorrect? 69
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 70
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 71
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 72
Proteins of SOS • Exactly how the SOS bypass system functions is not clear, although in E. coli it is known to be dependent on at least three genes, recA, umuC, and umuD • Current models for SOS bypass suggest that the UmuC and UmuD proteins combine with the polymerase III DNA replication complex 73
The mechanism • DNA polymerase III pauses at a type of damage called a TC photodimer • This region attracts single-strand- 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 74
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 75
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 76
MUTATORS 77
What is a mutator? • Mutant strains with increased spontaneous mutation rates have been detected • Such strains are termed mutators • In many cases, the mutator phenotype is due to a defective repair system • In humans, these repair defects often lead to serious diseases 78
Disease related to DNA repair Condition Sensitivity Cancer Mutated gene susceptibility Ataxia γ irradiation Lymphomas ATM (cell cycle and telangiectasia DNA repair) Bloom syndrome Mild alkylating Carcinomas, ATM (cell cycle and agents leukemias, DNA repair) lymphomas Cockayne syndrome UV light ERCC6 or ERCC8 (DNA repair) Fanconi anemia Cross-linking agents Leukemias Multiple (response to DNA damage) Xeroderma UV light, chemical Skin carcinomas Nucleotide excision pigmentosum mutagens and melanomas repair HNPCC Colon, ovary Mismatch repair 79
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 80
DNA sequencing and PCR
Resources • This lecture • Campbell and Farrell’s Biochemistry, pp. 377 -380, 384-387 82
What is DNA sequencing? • DNA sequencing is the process of determining the exact order of the chemical building blocks, that are the A, T, C, and G bases, that make up the genome 83
Importance of knowing the DNA sequence • Identification of genes and their localization • Identification of protein structure and function • Identification of DNA mutations • Genetic variations among individuals in health and disease • Prediction of disease-susceptibility and treatment efficiency • Evolutionary conservation among organisms 84
DNA sequencing of organism genome • Viruses and prokaryotes first • This was followed by the determination of the sequence of human mitochondrial DNA 85
Then simple eukaryotes • The first eukaryotic genome to be completely sequenced was that of yeast, Saccharomyces cerevisiae, which comprises approximately 12 million base pairs, distributed on 16 chromosomes • This achievement was followed by the first complete sequencing of the genome of a multicellular organism, the nematode Caenorhabditis elegans, which contains nearly 100 million base pairs 86
Last, but not least… • In humans, it is determining the sequence of the 3 billion bases • Determination of the base sequence in the human genome was initiated in 1990 and completed in May 2006 via the Human Genome Project • Achieving this goal has helped reveal the estimated 20,000-30,000 human genes within our DNA as well as the regions controlling them 87
Method of DNA sequencing • Based on premature termination of DNA synthesis by dideoxynucleotides 88
The process… • DNA synthesis is initiated from a primer that has been labeled with a radioisotope • Four separate reactions are run, each including deoxynucleotides plus one dideoxynucleotide (either A, C, G, or T) • Incorporation of a dideoxynucleotide stops further DNA synthesis because no 3 hydroxyl group is available for addition of the next nucleotide 89
Generation of fragments • A series of labeled DNA molecules are generated, each terminated by the dideoxynucleotide in each reaction • These fragments of DNA are then separated according to size by gel electrophoresis and detected by exposure of the gel to X-ray film • The size of each fragment is determined by its terminal dideoxynucleotide, so the DNA sequence corresponds to the order of fragments read from the gel 90
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Fluorescence-based DNA sequencing • Large-scale DNA sequencing is frequently performed using automated systems using fluorescence-based reactions using labeled ddNTPs • In this case, all four terminators can then be placed in a single tube, and only one reaction is necessary • The reactions are run into one lane on a gel and a machine is used to scan the lane with a laser 94
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Detection of fragments • The wavelength of fluorescence can be interpreted by the machine as an indication of which reaction (ddG, ddA, ddT, or ddC) the product came from • The fluorescence output is stored in the form of chromatograms 96
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Why is fluorescence better than radioactivity? • It eliminates the use of radioactive reagents • Can be readily automated 98
Polymerase Chain Reaction • In 1984, Kary Mullis devised a method called the polymerase chain reaction (PCR) for amplifying specific DNA sequences • PCR allows the DNA from a selected region of a genome to be amplified a billionfold, effectively "purifying" this DNA away from the remainder of the genome • The PCR method is extremely sensitive; it can detect a single DNA molecule in a sample 99
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