Can DNA Be Repaired?

A fundamental difference from RNA, protein, lipid, etc. • All these others can be replaced, but DNA must be preserved • Cells require a means for repair of missing, altered or incorrect bases, bulges due to insertion or deletion, UV-induced pyrimidine dimers, strand breaks or cross-links • Two principal mechanisms: mismatch repair and methods for reversing chemical damage
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Mismatch Repair
• Mismatch repair systems scan DNA duplexes for mismatched bases, excise the mispaired region and replace it • Methyl-directed pathway of E. coli is an example • Since methylation occurs post-replication, repair proteins identify methylated strand as parent, remove mismatched bases on other strand and replace them
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UVA irradiation causes dimerization of adjacent thymine bases. A cyclobutyl ring is formed between carbons 5 and 6 of the pyrimidine rings. Normal base pairing is disrupted by the presence of such dimers.

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Reversing Chemical Damage
• Pyrimidine dimers can be repaired by photolyase • Excision repair: DNA glycosylase removes damaged base, creating an "AP site" • AP endonuclease cleaves backbone, exonuclease removes several residues and gap is repaired by DNA polymerase and DNA ligase
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Base excision repair. A damaged base (■) is excised from the sugar-phosphate backbone by DNA glycosylase, creating an AP site. Then, an apurinic/apyrimidinic endonuclease severs the DNA strand, and an excision nuclease removes the AP site and several nucleotides. DNA polymerase I and DNA ligase then repair the gap.

Chemistry 40

Base excision repair. A damaged base (■) is excised from the sugar-phosphate backbone by DNA glycosylase, creating an AP site. Then, an apurinic/apyrimidinic endonuclease severs the DNA strand, and an excision nuclease removes the AP site and several nucleotides. DNA polymerase I and DNA ligase then repair the gap.

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Base excision repair. A damaged base (■) is excised from the sugar-phosphate backbone by DNA glycosylase, creating an AP site. Then, an apurinic/apyrimidinic endonuclease severs the DNA strand, and an excision nuclease removes the AP site and several nucleotides. DNA polymerase I and DNA ligase then repair the gap.

Chemistry 40

Base excision repair. A damaged base (■) is excised from the sugar-phosphate backbone by DNA glycosylase, creating an AP site. Then, an apurinic/apyrimidinic endonuclease severs the DNA strand, and an excision nuclease removes the AP site and several nucleotides. DNA polymerase I and DNA ligase then repair the gap.

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What Is the Molecular Basis of Mutation?
• Point mutations arise by inappropriate basepairing • Mutations can be caused by base analogs • Chemical mutagens react with bases in DNA • Insertions and deletions

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MUTANT X

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transition transversion

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Point mutations due to base mispairings. (a) An example based on tautomeric properties. The rare imino tautomer of adenine base pairs cytosine rather than thymine. (1) The normal A-T base pair. (2) The A*-C base pair is possible for the adenine tautomer in which a proton has been transferred from the 6-NH2 of adenine to N-1. (3) Pairing of C with the imino tautomer of A (A*) leads to a transition mutation (A-T to G-C) appearing in the next generation. (b) A in the syn conformation pairing with G (G is in the usual anti conformation). (c) T and C form a base pair by H-bonding interactions medicated by a water molecule.

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DNA Techniques

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Agarose Gel Electrophoresis

technique used in the laboratory that results in the separation of charged molecules

Chemistry 40

In the case of DNA we can separate the molecules based on their size. DNA has a negative charge in solution, so it will migrate to the positive pole in an electric field.
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Southern Blotting
developed in l975 by Edward M. Southern at the University of Edinburgh makes use of radiolabelled probes that will hybridize with a specific sequence of DNA

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DNA Markers
SNP- single nucleotide polymorphism most frequent RFLP- restriction fragment length polymorphism RAPD – random amplified polymorphism SSCP- single stranded chain conformation polymorphism STD- short tandem DNA
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DNA Markers
Mitochondrial DNA Analysis older biological samples that lack nucleated cellular material, such as hair, bones, and teeth, cannot be analyzed with STR and RFLP Y-Chromosome Analysis especially useful for tracing relationships among males or for analyzing biological evidence involving multiple male contributors
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Northern Blotting
detection of specific RNA sequences. developed by James Alwine and George Stark at Stanford University and was named such by analogy to Southern blotting.

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Western Blotting
allows investigators to determine, with a specific primary antibody, the relative amounts of the protein present in different samples

Chemistry 40

What Does It Mean: “To Cloning”?
Clone: a collection of molecules or cells, all identical to an original molecule or cell • To "clone a gene" is to make many copies of it - for example, in a population of bacteria • Gene can be an exact copy of a natural gene • Gene can be an altered version of a natural gene • Recombinant DNA technology makes it possible
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Plasmids
Naturally occurring extrachromosomal DNA • Plasmids are circular dsDNA • Plasmids can be cleaved by restriction enzymes, leaving sticky ends • Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid
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One of the first widely used cloning vectors, the plasmid pBR322. This 4363-bp plasmid contains an origin of replication (ori) and genes encoding resistance to the drugs ampicillin (ampr)and Tetracycline (tetr). The locations of restriction endonuclease cleavage sites are indicated.

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Chimeric Plasmids
Named for mythological beasts with body parts from several creatures • After cleavage of a plasmid with a restriction enzyme, a foreign DNA fragment can be inserted • Ends of the plasmid/fragment are closed to form a "recombinant plasmid" • Plasmid can replicate when placed in a suitable bacterial host
Chemistry 40

Restriction endonuclease EcoRI cleaves doublestranded DNA. The recognition site for EcoRI is the hexameric sequence GAATTC: 5' . . . NpNpNpNpGpApApTpTpCpNpNpNpNp . . . 3' 3' . . . NpNpNpNpCpTpTpApApGpNpNpNpNp . . . 5' Cleavage occurs at the G residue on each strand so that the DNA is cut in a staggered fashion, leaving 5'overhanging single-stranded ends (sticky ends): 5' . . . NpNpNpNpG pApApTpTpCpNpNpNpNp . . . 3' 3' . . . NpNpNpNpCpTpTpApAp GpNpNpNpNp . . . 5' An EcoRI restriction fragment of foreign DNA can be inserted into a plasmid having an EcoRI cloning site by (a) cutting the plasmid at this site with EcoRI , annealing the linearized plasmid with the EcoRI foreign DNA fragment, and (b) sealing the nicks with DNA ligase .

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What Is the Polymerase Chain Reaction (PCR)?
What if you don't have enough DNA for colony hybridization or Southern blots? • The small sample of DNA serves as template for DNA polymerase • Make complementary primers • Add primers in more than 1000-fold excess • Heat to make ssDNA, then cool • Run DNA polymerase (usually Taq) • Repeat heating, cooling, polymerase cycle
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Chemistry 40 Fig. 10-1, p.241

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