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IUB BIOL-L 211 - Double-Stranded Breaks and Repair Mechanisms

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BIOL-L211 Lecture 14 Outline of Last Lecture I. MutagensII. Base Excision RepairIII. Nucleotide Excision RepairIV. Error Prone PolymeraseV. DNA PhotolyaseVI. Methyltransferase Outline of Current Lecture I. Double-Stranded BreaksII. Repair Mechanisms for DSBsIII. ArticleCurrent LectureDouble-stranded Breaks, Non-Homologous End Joining, and Homologous RecombinationI. Double-Stranded Breaks (DSBs)A. DSBs: one of the most dangerous forms of DNA damage due to the breaking of the phosphodiester bond, which can result in deletions1. Note that phosphodiester bonds are what keep strands togetherB. Causes of DSBs1. Radiation (such as UV radiation)2. Physical constraints (where DNA tangles/bends and ultimately fractures)3. Reactive oxygen species (oxygen actually has very destructive potential)a. Chemically reactive molecules with oxygen in them (superoxide)b. Products of metabolism of oxygen, immune cells, and radiationThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.c. Superoxide reacts with and damages all macromolecules with which it interacts (especially DNA- modifying bases or producing DSBs)II. Repair Mechanisms for DSBsA. Homologous Recombination (HR)1.Homologous chromosomes are used as repair templatea. Two homologous DNA molecules alignb. Resection: short stretch of DNA digested to yield ssDNA with 3' tailsc. Strand Invasion: ssDNA tail invades unbroken chromosomed. Strand pairing: Invading strand base pairs with its complementary strande. DNA synthesis then uses complementary strands as templatesf. Strands cross, connecting the DNA molecules; Holliday junctions are formed, which must be resolvedi. Resolved through cleavages in both junctionsii. Strand can return to original position, but crossovers may resultiii. Be able to determine specific product from one Holliday Junction cleavage2. Most common pathway for DSB repair3. In Bacteria, which have only one chromosome, template can come from environmenta. Transformation: process by which some bacteria incorporate DNA from environment (derived from Transforming Principle)i. dsDNA bound by com machinery (see below)ii. dsDNA proteolyzed, yielding ssDNAiii. ssDNA assimilated by cell (can fix DSBs)b. Competent bacteria are capable of transformationc. Competence is the physiological state in which bacteria take up DNA (Different from competent- adjective vs. noun)d. Com machinery: composed of over 40 proteins in the bacterial membrane that help import DNA from the environmentB. Non-Homologous End Joining (NHEJ)1. Reunites broken ends of DNAa. Ku70 and Ku80 form heterodimer on broken endsb. Ends brought together by microhomology, or the fact that the sequences are similarc. End processing: 5' and 3' overhangs removed (mutagenic)d. Template completed2. Generally backup DSB repair systemIII. Article: "DNA May Have Had Humble Beginnings as Nutrient Carrier"- Astrobiology Magazine, Sept. 2014A. Background1. Generally bacteria and archaea have only one chromosome2. Multiple chromosomes are beneficial because they are good backup and assistin recombination3. Haloferax volcanii (archaea) comes from the dead sea, and its number of chromosomes varies depending on its environment (from 10-20)B. Experiment1. Experiment showed that in environments with limited phosphate, Haloferax volcanii possessed fewer chromosomes, yet cells continued to grow and divide (ribosome count was constant)2. Researchers concluded that H. volcanii destroyed (cannibalized) its own chromosomes to obtain the phosphate lacking in the


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