BBMB 405 1st Edition Lecture 27Outline of Last Lecture XIV. Chapter 28: DNA Replication, Repair, and RecombinationD. Many types of DNA damage can be repairedOutline of Current Lecture XIV. Chapter 28: DNA Replication, Repair, and RecombinationE. DNA recombination plays important roles in replication, repair, and other processesXV. Chapter 29: RNA Synthesis and ProcessingA. ReviewB. RNA Polymerases catalyze transcriptionCurrent LectureXIV. Chapter 28: DNA replication, repair, and RecombinationE. DNA recombination plays important roles in replication, repair, and other processes1. Recombinationa. Creates genetic diversityb. Exchange of genetic information between two different pieces of DNA- Share same gene with each other- Reciprical recombination: both receive DNA from the otherc. Essential in these processes- Restart stalled replication process- Repair double-stranded breaks- Provides diversity by exchange of DNA during meiosis- Generates diversity in antibodies- Integrates genetic material of some viruses into host’s DNA- Use for generation of “knockout” miceThese 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.d.2. Strand invasion/RecAa.b. Synapse: DNA is brought close to each otherc. RecA: Forms nucleoprotein filaments, opens origin of replication, attaches to pink,blue, and purple strands3. Double strand break repair: occurs because of error in replicationa. Homologous recombination (HR): use sister chromosome to repair break- Accurate repair, can introduce specific sequence changes present in homologous DNA- Recognize: recession of 5’ end on both sides, then one undergoes strand invasion, repair, then serve as template for other strand- Occurs at same time as replication, but not during G1b. Non-homologous end joining (NHEJ): backup, used in G1- Error prone: leads to indel (random insertion or deletion)- Has no template so error prone DNA repair mechanism that can add or delete base pairs4. Genetic Editinga. Engineer genome by double strand breaks: - Find gene of interest and target- Results in indels- Need a frameshift of anything but 3- Can also introduce separate DNA that contains mutationb. Process- Site-specific endonuclease designed to cleave gene of interest- Double strand break introduced by endonuclease cleavage- NHEJ or HR repair leads to mutation: NHEJ creates random mutations (indels), HR makes specific mutation and requires introduction of homologous DNA5. CRISPR-Cas9: able to site specific sequences and target certain genes, programmable RNA guided site-specific endonuclease6. Holiday Junctions:a. Crerecombinase: circularizes genome, covalent 3’-phosphotyrosine intermediateb.7. NExtXV. Chapter 29: RNA Synthesis and ProcessingA. Review1. RNA and roles in gene expressiona. Replication: priming RNA, telomerase RNAb. Transcription: riboswitch, long noncoding RNAc. Translation: ribosomal RNA, transfer RNA, signal recognition particle RNA, transfer-messenger RNAd. Gene regulation: small interfering RNA, micro RNAe. Processing: small nuclear RNA, small nucleolar RNA, ribonuclease P, self-splicing intronsf. Genome integrity: Piwi interacting RNA, retrotransposonsg. Other: RNA genomes, CRISPR RNA, circular RNA2. RNA are processed post-transcription: Splicing, chemical modification, addition, trimming, cleavage, editing3. RNA transcription is catalyzed by RNA polymerase; hydrolysis of NTP provides energy for creation of phosphodiester bond formationB. RNA polymerases catalyze transcription1. Hydrolysis of NTP provides energy for creation of phosphodiester bond formation2. Initiation: transcription bubble unwinds 17 base pairs and maintains by reanealing base pairs already transcribed3. Elongation: fidelity not as high, don’t need accuracy level of DNA, once fall off it can’t restart, polymerase needs to get through entire sequence4. Subunits of RNAP and function (in bacteria, eukaryotes are more complex as always)a.b. alpha, beta, beta prime and omega are core subunits in that they stay part of the complex through out all of transcriptionc. Alpha: RNAP assembly, interaction with promoter DNA, interactions with regulatory factorsd. Beta and beta prime: active site, interactions with DNA/RNAe. Omega: stabilizes assembled RNAPf. Sigma: transcription initiation, part of RNAP holoenzyme, but dissociates during elongation5. RNA polymerase uses two divalent metal ions, similar to DNA polymerase in that ther is active nucleotide and stabilizes leaving group6. DNA is unwound locally at transcription bubblea. DNA template bases exposed within bubbleb. DNA duplex re-anneals as template moves out of bubblec. Nascent RNA is separate from templated. Elongation occurs at 50
View Full Document
Unlocking...