MBIO 3410 lecture Sept 27 2007

MBIO 3410 lecture Sept 27 2007 - Last lecture Mutagenesis...

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1 Last lecture Mutagenesis Physical Mutagens Chemical mutagens DNA damage DNA lesions Oxidative damage Alkylation Bulky adducts DNA repair Nucleotide excision repair (NER) Base excision repair (BER) Mismatch repair DNA recombination Homologous recombination Homologous recombination in E. coli Site-specific recombination E. coli- Bacteriophage lambda Site-specific recombination in eukaryotes Transposition Prokaryote transposons Eukaryotic transposons Restriction-modification systems Methylase modification Restriction endonucleases (restriction enzymes) Types of restriction modification systems
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2 Types of restriction modification systems Classification based on: Enzyme complexity Cofactor requirements Position of DNA cleavage
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3 Type I Recognizes a DNA sequence but cleaves in an apparent random fashion away from the recognition site Requires ATP, S-adenosylmethionine (SAM) and magnesium ions (Mg 2+ ) for activity Composed of three subunits: Specificity subunit for DNA recognition site Modification subunit Restriction subunit In the presence of SAM the enzyme binds the recognition site If methylated - then ATP hydrolysis stimulates the dissociation of the enzyme from DNA If hemimethylated - ATP methylates of the other strand If unmethylated - then DNA cleavage occurs
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4 Type II They recognize symmetrical DNA sequences where the 5’ to 3’ direction on one DNA strand is the same as the sequence in the 5’ to 3’ direction ( palindrome) Require Mg 2+ for activity cleave DNA within the recognition site Different restriction enzymes isolated from different sources often recognize the same DNA sequence, although they may cleave the DNA differently ( isoschizomer) Sma I and Xma I both recognize the same DNA sequence 5’-CCCGGG-3’ 3’-GGGCCC-5’ Sma I cleaves in the middle of the site between the central C and G nucleotides while Xma I cleaves after the first C nucleotide
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5 Type II restriction enzyme activity Little sequence homology at the amino acid level Conversed catalytic core composed of a five-stranded β - sheet flanked by two α -helices Function by ‘scanning’ the length of a DNA molecule by binding to it in a non-specific fashion Recognition of a specific sequence causes the restriction enzyme to undergoes a large conformational change activating the catalytic sites cutting the two sugar–phosphate backbones of the DNA double helix to generate a 3’ hydroxyl and a 5’ phosphate Bam HI
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6 Type III The type III restriction systems act as complexes of two different subunits: subunit (M) is responsible for DNA sequence recognition and modification subunit (R) is responsible for nuclease action DNA cleavage requires Mg 2+ and SAM The recognition sites are approximately symmetric, and cleavage occurs by nicking one DNA strand at a measured
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