Midterm 2 concepts

Midterm 2 concepts - Midterm 2 concepts: DNA REPLICATION...

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Midterm 2 concepts: DNA REPLICATION Models for DNA Replication: 1. Conservative 2. Semi-Conservative Ways to distinguish between old and new DNA strand: 1. CsCl gradient to separate DNA with a different isotope of nitrogen (N14=light, N15=heavy) 2. BrdU (Bromine labeled Thymine gets incorporated into new DNA in place of T)-Two strands are light and one strand is dark when stained with a combination of fluorescent dye and Giemsa stain DNA Polymerase 1. Needs primer (has to be 2 nucleotides or longer) with free 3’OH, and DNA template 2. Synthesizes DNA 5’-3’ 3. Adds triphosphate nucleotide when 3’OH attacks incoming alpha phosphate and two phosphates are removed 4. Arg and Lys (+charged amino acids) hold the phosphates from the incoming nucleotide in close proximity to the 3’OH on primer 5. Tyrosine (has aromatic ring) makes base stacking interaction with bases in the DNA also facilitating the addition of a new nucleotide A. Polymerase III 1. 5’-3’ DNA polymerase ; main polymerase synthesizing DNA of lagging and leading strand 2. 3’-5’ exonuclease activity involved in proofreading 3. 2 subunits-synthesize new DNA using both template strands at same time 4. CANNOT synthesize primers, remove primers, or ligate DNA B. Polymerase I 1. Removes RNA primer ( 5’-3’ exonuclease ) and DNA ( 3’-5’ exonucelase - proofreading) 2. CANNOT ligate DNA 3. 5’-3’ DNA polymerase DNA polymerase III holoenzyme Components: 1. Sliding Clamp (B subunit) –holds DNA polymerase to DNA 2. Helicase -unwinds the DNA; requires ATP 3. Single Stranded Binding Proteins -stabilize single stranded DNA and prevents its degradation by nucleases 4. Topoisomerases -breakage and joining of DNA to relieve supercoiling 5. Primase -makes RNA primer 6. Tau -holds the two subunits of polymerase together 7. Initiator -known in prokaryotes to recognize and bind specific DNA sequences at
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origin or replication 8. DNA ligase -ligates two nucleotides together in DNA backbone-coupled to ATP hydrolysis **Know why the leading strand is synthesized continuously and the lagging strand is synthesized in small fragments each with their own primers **Be able to draw a replication fork with the direction of synthesis of the new strands and which of the strands will have Okazaki fragments (discontinuous) and which will be continuous, also know where the primers will be DNA Synthesis is… 1. Semi-conservative 2. Bidirectional (2 forks at every origin of replication) 3. Continuous (leading strand) 4. Discontinuous (lagging strand-okazaki fragments) “End replication problem ”- When the primer at the end of the 5’ end of the lagging strand is removed by Pol I no new DNA is synthesized there, if this were to occur every round of replication your chromosomes will continue to get shorter The enzyme telomerase adds a repetitive sequence of DNA (using an internal RNA as
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This note was uploaded on 07/08/2009 for the course LIFESCI LS 3 taught by Professor Paulolague during the Fall '09 term at UCLA.

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Midterm 2 concepts - Midterm 2 concepts: DNA REPLICATION...

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