BCHM 4116 1st Edition Exam 2 Study Guide Lectures 13 22 Lecture 13 I DNA replication a DNA polymerase selects base b Nucleophilic attack by 3 OH on alpha phosphate of incoming dNTP c New phosphoester bond formed d Replicates in 5 to 3 e Release of PPi II DNA replication is a Semi conservative i 1 of 2 original strands is conserved in each progeny molecule b Bi directional i Replication goes both directions ii Helicase 1 Unwinds double helices 2 ATP dependent iii DNA gyrase 1 AKA topoisomerase 2 Overcomes torsional stress by phosphodiester bond breakage and reunion c Semi discontinuous III Requirements of DNA synthesis a Template b Primer w 3 OH c dNTPs d DNA polymerase e Accessory proteins Lecture 14 1 How is DNA Replicated a Semiconservative i 1 of 2 original strands are conserved in each of the 2 progeny b Bidirectional i 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 gyrase 1 Type II topoisomerase 2 Overcome torsional stress imposed upon unwinding by introducing negative supercoils ii Helicase 1 Unwinds 2 Disrupt hydrogen bonds iii Single stranded DNA binding proteins 1 Binds to single strands to prevent from re annealing iv DNA Polymerase 1 Uses ssDNA as template 2 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 ligase 1 Seals remaining nicks in Okazaki fragments vii Ter Region 1 DNA replication terminates Ter 2 Termination requires binding of Tusprotein toTer a Tus is a counterhelicase i Tus prevents DNA duplex from unwinding by blocking progression of replication fork Lecture 15 1 Functions of DNA Polymerase a Synthesize DNA replication b NEEDS i Deoxynucleoside 5 triphosphateds dNTPs ii Primers 1 b c DNA pol can only elongate and can t join 2 dNTPs 2 must have free 3 OH end for DNA pol to add 3 RNA bases make up the primer 4 Primase synthesizes RNA primer on lagging strand a Leading strand only needs priming once at start c DNA pol I II V DNA repair d DNA Pol III DNA replicating enzyme for E coli i Auxiliary subunits increase polymerase activity and its processivity 2 DNA Replicated in Eukaryote Cells a 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 replication b Cell cycle controls timing of DNA replication i Checkpoints control whether cell continues to next phase ii Checkpoints depend on cyclins and cyclin dependent protein kinases 1 Cyclin class of proteins synthesized at 1 phase of cell cycle and degraded at another c Initiation of replication i 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 16 1 Replication of the Ends of Chromosomes a Repeated G C rich sequences at ends of chromosome b Telomeres protect against DNA degradation c Telomerase adds telomers d Telomerase is a specialized reverse transcriptase containing catalytic subunit e Telomerase uses 3 end of DNA as primer and adds TTAGGG repeats 2 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 intermediate Lecture 17 1 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 intermediate d The 3 Enzymatic Activities of RT i RNA directed DNA polymerase activity ii 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 duplex e RT i Enters host cell ii Uses host DNA nucleotides to convert viral ssRNA to ssDNA 1 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 recombination i Exchange of DNA sequences b w homologous chrom resulting in arrangement of genes into new combo b Non homologous recombination i 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 19 1 Recombination a RecBCDEnezyme Complex Unwinds dsDNA and Cleaves its single strands i Fig 28 19 ii RecBCD 1 Unwinds dsDNA 2 Rec B Rec D helicases 3 Rec B nuclease domain nuclease activity cleaves both of the newly formed single strands 4 SSB binds to single strand iii 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 Recombinase 2 Can bind ssDNA and interact with dsDNA v Ruv A B C 1 Holiday junction 2 A B 3 Ruv C cleaves a Endonuclease that resolves Holiday junction into heteroduplex recombination products Lecture 20 1 DNA Repair a Human genome has 150 genes associated w DNA repair b DNA repair system includes i Direct reversal damage repair 1 Chem Rxn that reverse damage returning DNA to proper state ii Single strand damage repair 1 Relies on complementary strand to guide repair 2 Mismatch repair MMR a Mismatched during replication b Scans excises mismatched region replaces c KEY to tell which strand is newly synthesized and thus contains mismatch is via METHYLATION i Newly synthesized strands are not yet methylated whereas parent strand is d Endonuclease cuts new unmethylated strand