BCHM 4116 1st Edition Exam# 2 Study Guide Lectures: 13 - 22Lecture 13I. DNA replication a. DNA polymerase selects base b. Nucleophilic attack by 3’-OH on alpha phosphate of incoming dNTPc. New phosphoester bond formed d. Replicates in 5’ to 3’e. Release of PPiII. DNA replication is: a. Semi-conservativei. 1 of 2 original strands is conserved in each progeny molecule b. Bi-directional i. Replication goes both directionsii. Helicase1. Unwinds double helices2. ATP-dependent iii. DNA gyrase1. AKA topoisomerase2. Overcomes torsional stress by phosphodiester bond breakage and reunion c. Semi-discontinuous III. Requirements of DNA synthesisa. Templateb. Primer w/ 3’ OHc. dNTPsd. DNA polymerasee. Accessory proteins Lecture 141. How is DNA Replicated? a. Semiconservativei. 1 of 2 original strands are conserved in each of the 2 progeny b. Bidirectionali. Begins at 1 or more specific region called origin of replication ii. Involves 2 replication forks that move in opposite directions c. Semi-discontinuous i. Continuous strand = Leading strand ii. Discontinuous strand = Lagging strand via Okazaki fragments iii.d. Replication:i. DNA gyrase1. Type II topoisomerase2. Overcome torsional stress imposed upon unwinding by introducing negative supercoils ii. Helicase1. Unwinds 2. Disrupt hydrogen bonds iii. Single-stranded DNA binding proteins 1. Binds to single strands to prevent from re-annealing iv. DNA Polymerase1. Uses ssDNA as template2. Synthesize DNA only in 5’3’ v. DNA Pol I 1. Only Pol with 5’3’ exonuclease, meaning it can replace RNA primer with DNA vi. DNA ligase1. Seals remaining nicks in Okazaki fragments vii. Ter Region1. DNA replication terminates @ Ter2. Termination requires binding of Tusprotein toTera. Tus is a counterhelicasei. Tus prevents DNA duplex from unwinding by blocking progression of replication fork Lecture 151. Functions of DNA Polymerasea. Synthesize DNA replicationb. NEEDS: i. Deoxynucleoside-5’-triphosphateds (dNTPs)ii. Primers1. b/c DNA pol can only elongate and can’t join 2 dNTPs2. must have free 3’OH end for DNA pol to add 3. RNA bases make up the primer4. Primase synthesizes RNA primer on lagging stranda. Leading strand only needs priming once at start c. DNA pol I, II, V = DNA repaird. DNA Pol III = DNA replicating enzyme for E. coli i. Auxiliary subunits increase polymerase activity and its processivity2. DNA Replicated in Eukaryote Cellsa. Eukaryotic cells have to replicate their enormous genomes in the few hours allotted to the S phase by initiating DNA replication at multiple origins of replicationb. Cell cycle controls timing of DNA replicationi. Checkpoints control whether cell continues to next phaseii. Checkpoints depend on cyclins and cyclin-dependent protein kinases 1. Cyclin = class of proteins synthesized at 1 phase of cell cycle and degraded atanother c. Initiation of replicationi. Depends on origin recognition complex (ORC) 1. Protein complex that binds to replication origins ii. Demands DNA replication occurs only once per cell cycle Lecture 161. Replication of the Ends of Chromosomes a. Repeated G-C rich sequences at ends of chromosomeb. Telomeres protect against DNA degradation c. Telomerase adds telomersd. Telomerase is a specialized reverse transcriptase containing catalytic subunit e. Telomerase uses 3’ end of DNA as primer and adds TTAGGG repeats2. Replication of Genomes a. Viruses have genomes composed of RNA not DNA, so how is RNA replicated? b. Reverse Transcriptase (RT): RNA-directed DNA polymerase i. RT synthesizes in 5’  3’ ii. Requires primer 1. Primer = specific tRNA molecule captured by virion of host cell c. Retroviruses: RNA viruses that replicate their RNA genomes via a DNA intermediateLecture 171. Replication of Genomes a. Viruses have genomes composed of RNA not DNA, so how is RNA replicated? b. Reverse Transcriptase (RT): RNA-directed DNA polymerase i. RT synthesizes in 5’  3’ ii. Requires primer 1. Primer = specific tRNA molecule captured by virion of host cell c. Retroviruses: RNA viruses that replicate their RNA genomes via a DNA intermediated. The 3 Enzymatic Activities of RTi. RNA – directed DNA polymerase activityii. RNase H activity 1. RNase H is a nuclease that degrades RNA chains in DNA: RNA hybrid 2. Degrades template genomic RNA &tRNA after DNA synthesis is completed iii. DNA- directed DNA polymerase activity 1. Replicates ssDNA remaining after RNase H degradation of viral genome, yielding DNA duplexe. RTi. Enters host cellii. Uses host DNA nucleotides to convert viral ssRNA to ssDNA1. This synthesized ssDNA has lots of random errors b/c poor proof reading activity iii. ssDNA is then again reverse transcribed into dsDNA via RT iv. integrase an enzyme that came with the virus takes the dsDNA into the nucleus of the host cell & integrates the viral dsDNA with the host DNA Lecture 18 1. Recombination a. Homologous recombinationi. Exchange of DNA sequences b/w homologous chrom., resulting in arrangement of genes into new combo. b. Non-homologous recombinationi. Different nucleotide sequences recombine c. Transposition i. Enzymatic insertion of a transposon (mobile segment of DNA) into new location in genome d. Homologous Recombination Proceeds according to Holiday Model i. Chromosome pairing is called synapsis ii. Begins w/ introduction of single stranded nicks in DNA iii. 2 duplex partially unwind iv. free single stranded end of 1 duplex begins to base-pair w/ nearby complementary Lecture 191. Recombination a. RecBCDEnezyme Complex Unwinds dsDNA and Cleaves its single strands i. Fig. 28.19 ii. RecBCD1. Unwinds dsDNA2. Rec B & Rec D = helicases3. Rec B = nuclease domain, nuclease activity cleaves both of the newly formedsingle strands4. SSB binds to single strandiii. Chi site (5’ – GCTGGTGG – 3’)1. RecBCD stops cutting at 3’ but cont. at 5’ iv. Rec A binds to 3’ to form nucleoprotein filament 1. AKA Recombinase2. Can bind ssDNA and interact with dsDNAv. Ruv A. B. C 1. Holiday junction2. A & B 3. Ruv C = cleavesa. Endonuclease that resolves Holiday junction into heteroduplex recombination productsLecture 20 1. DNA Repaira. Human genome has 150 genes associated w/ DNA repair b. DNA repair system includes:i. Direct reversal damage repair1. Chem. Rxn that reverse damage, returning DNA to proper stateii. Single-strand damage repair1. Relies on complementary strand to guide