Molec Bio Exam 3 – Flashcards
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| Cell Cycle Phases |
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| • G1: checkpoint 1 • G2: checkpoint 2 • S: synthesis phase • M: mitosis phase (cell division) *cell cycle will stop if any issues w/ 1st or 2nd checkpoints |
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| S Phase |
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| • synthesis phase • DNA replication (each DNA molecule = chromatid) • sister chromatid adhesion • in bacteria, replication & segregation occur in same step |
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| Mitosis |
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| • cell division where parental cell is used to form two identical daughter cells • chromosome segregation • sister chromatids segregated to opposite poles of cell; each daughter cell thus rcvs identical info when cell divides • Phases: PMATC (prophase, metaphase, anaphase, telophase) • cytokinesis actually splits one cell into two |
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| G1 |
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| • first checkpoint • makes sure DNA not damaged before S phase • parental cell preparing for DNA replication |
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| G2 |
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| • 2nd checkpoint • makes sure DNA fully replicated before M phase • cell preparing for chromosome segregation |
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| 2 major events during cell cycle |
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| • DNA replication • chromosome segregation |
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| What happens during checkpoints - G1 & G2 |
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| • allow cells to check to ensure necessary materials and energy present to proceed to next phase • makes sure DNA not damaged |
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| sister chromatids |
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| pair of DNA duplicated molecules |
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| chromatid |
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| individually copied DNA molecule |
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| sister chromatid cohesion |
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| cohesin holds together sister chromatids until segregation |
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| microtubule-organizing centers |
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| • centrosomes (animal cell) or spindle pole bodies (yeast/fung) • form poles at opposite ends of cells • microtubules pull sister chromatids towards poles, preparing for cell division |
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| sister chromatid separation |
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| cohesion ring destroyed, sister chromatids segregate to opposite poles of cells |
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| Interphase |
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| • G1, S & G2 phases • when mitotic division not occuring • DNA is not compact or condensed |
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| Prophase |
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| • long DNA strands wrap into highly condensed chromosomes • nuclear envelope breaks down • microtubule organizing centers migrate to opposite poles of cell; signals metaphase |
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| Metaphase |
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| • microtubules (long fibers) attach to each kinetochore of chromosome pair (centromeres when chromatids) to organizing centers on either side • two chromosomes form bivalent attachment • microtubules pull sister chromatids towards each pole • chromosomes align in middle of cell because cohesin rings still surrounding them |
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| bivalent attachment (metaphase) |
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| • two kinetochores of sister chromatid pair are attached to microtubules of opposite sides (poles) of cell |
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| monovalent attachment (metaphase) |
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| • only one kinetochore of sister chromatid pair is attached to a microtubule on one side of cell (pole) • results in uneven segregation of chromosomes into daughter cells |
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| cohesin removed by… |
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| proteolytic destruction ® signals anaphase |
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| anaphase |
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| • cohesion between sister chromatids lost (via proteolysis) • chromosomes migrate to opposite poles of cell |
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| telophase |
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| • new nuclear membranes form around each sets chromosomes before cells split, final step mitosis |
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| cytokinesis |
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| cell divides into two daughter cells |
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| cell cannot perform cytokinesis |
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| one cell will have two nuclei with duplicate chromosomes |
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| cell cannot break apart cohesin rings |
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| anaphase affected and cell could not segregate chromosomes to opposite poles from center |
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| histones |
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| proteins that DNA winds around before tightly compacting |
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| nucleosomes |
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| • assembly to form compact DNA to fit into body • histone forms a spool with DNA wrapped around it • 8 histones (two each of H2A, H3, H2B & H4) form 1 core • assembled from histone protein subassemblies • H3 & H4 form tetramer subassembly • 2 H2A & H2B dimers form two subassemblies • each histone protein has N-terminal tail • DNA wraps around each histone core 1.65 times (147bp in length) |
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| human genome contains… |
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| • 3billion bp = 1 meter long if stretched out • to fit into body, DNA wound into 23 pairs of chromosomes |
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| when cell divides… |
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| • DNA must be duplicated • two new daughter strands rapidly reassembled into nucleosomes • old histones distributed between two new DNA strands • new histones brought in to fill in gaps |
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| N-terminal tail on histone |
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| • provide guide for DNA to wrap around histone core • emerge between DNA strands & create groove (like screw) • force DNA to twist around histone in left-handed manner |
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| what binds DNA to core of histone |
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| via hydrogen bonds |
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| H3 & H4 tetramer binds what part of DNA? |
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| middle & both ends of core DNA |
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| H2A and H2B dimers bind to what part of DNA? |
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| binds DNA between middle and ends |
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| how are multiple nucleosomes connected? |
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| • adjacent nucleosomes connected by short stretches of DNA called linker DNA • fifth histone, H1 protein, is linker histone that binds linker DNA |
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| H1 linker protein |
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| • stimulates first level of chromatin packing, formation of 30nm fiber • binds both end linker DNA and middle of nucleosomal DNA, bringing adjacent nucleosomes into close proximity • larger than other histones at ~21 kD |
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| 10nm fiber |
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| • less condensed form of chromatin • "beads on a string" |
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| 30nm fiber |
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| • more condensed version of chromatin • one of two structures (might differ between species) ® • solenoid • zig-zag |
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| solenoid structure |
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| • nucleosome disks stacked on top of one another; forming helix; linker DNA packed inside |
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| zig-zag structure |
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| • requires longer linker DNA because of how the nucleosomes cross over one another from opposite sides (may be preferred form for species w/ longer linker DNA) |
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| highly condensed mitotic chromosome |
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| • 30nm fiber must fold even further • fold into large loops, held together by nuclear scaffolding proteins at the base of each loop • then folded again to form condensed chromosome |
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| chromosome duplication |
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| • associated proteins must be reassembled on each daughter DNA molecule • nucleosomes must be partially dissassembled to allow replication machinery to pass • histone synthesis & modification occurs |
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| histone synthesis & modification |
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| • H3:H4 tetramers remain bound to one of two daughter DNA duplexes at random • H2A:H2B intact but released into pool of free dimers, surrounding replication fork • H3:H4 tetramers from old nucleosomes form new nucleosomes on daughter DNA after replication fork passes • because amt of DNA doubled, more histones synthesized and recruited • histone chaparones guide to DNA behind replication fork |
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| histone chaparones |
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| • Asf1 & CAF-1 work together to bring new H3:H4 tetramer to assembly site that didn't get old tetramer, where PCNA ring is • NAP-1 brings two H2A:H2B dimers to each new strand (each gets one old dimer and one new dimer) • histone modifications carefully preserved • essential to survival of each daughter cell |
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| PCNA ring |
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| • proliferating cell nuclear antigen rings • sliding clamp proteins that tether DNAP to DNA during replication; left on new DNA to serve as markers |
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| modified old ; new histones |
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| • modified old histones can recruit enzymes that modify nearby new histones in same way • i.e. enzymes w/ bromodomains bind to acetylated histones ; acetylate nearby new histones |
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| Why is packing DNA into chromosomes important? |
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| • it allows DNA to fit into cell • protects DNA from damage • only packaged DNA can be transmitted to daughter cells • chromosomal DNA very stable (naked DNA not) • chromosome has overall organization to each DNA molecule |
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| Why is organization of DNA important? |
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| • organization regulates: gene expression ; recombination • recombination btw parental chromosomes - generates diversity seen w/ different individuals of any organism |
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| molecular mass of eukaryotic chromosome |
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| • half of mass is made up of proteins • most are histones and some are nonhistones |
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| chromatin |
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| • given region of DNA w/ its associated proteins • these proteins help to compact DNA |
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| histones |
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| • majority of DNA-associated proteins • small, basic proteins • have high contect of (+) charged amino acids • ; 20% of residues in histones are lysine or arginine |
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| nonhistone proteins |
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| • include DNA-binding proteins • they regulate transcription, regulation, repair ; recombination of cellular DNA |
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| size properties of chromosomes |
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| • human cell contains 3x109bp per haploid chromosomes set • avg thickness of each bp = 3.4A • if stretched out = ~1010A or 1m • diploid stretched out = 2m |
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| prokaryotic nucleoid |
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| • typically only has one complete copy of chromosome packaged into nucleoid • portions of chromosome present in two and sometimes four copies during rapid division |
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| plasmids |
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| • independent, circular DNA • not essential for bacterial growth • carry genes that confer desirable traits to bacteria (antibiotic resistance, etc) |
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| diploid |
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| • two copies of each chromosome • most eukaryotic cells are diploid |
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| homologs |
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| two copies of given chromosome (one from each parent) |
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| haploid |
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| • only single copy of each chromosome • involved in sexual reproduction |
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| polyploid |
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| • more than two copies of each chromosome • some organisms - majority of adult cells in polyploid state |
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| global genome amplification |
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| • hundreds or thousands of copies of each chromosome • allows cell to generate larger amts of RNA ; proteins |
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| megakaryocytes |
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| • specialized polyploid cells • produce thousands platelets that lack chromosomes but essential to human blood (maintains high metabolism) |
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| nucleus |
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| chromosomes contained w/i membrane-bound organelle |
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| genome size, number of genes |
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| • correlates with organism's complexity • generally though, it's the number of genes, not necessarily genome size • prokaryotic cells have genomes ;10Mb • single-cell eukaryotes ;50Mb • complex protozoans ;200Mb |
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| genome density |
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| increased complexity = less gene density |
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| intergenic sequences |
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| • decrease gene density • discontinuous, protein-coding regions • takes up more than 60% of human genome • either unique or repeated |
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| introns |
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| • interspersed non-protein-coding regions • removed from RNA after transcription • 95% of average protein-coding gene (5% actually encodes) |
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| RNA splicing |
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| removes introns from RNA before translation = mature mRNA |
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| unique intergenic DNA |
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| • 25% of intergenic DNA • regions of DNA required to direct/regulate transcription • nonfunct relics, mutant genes, fragments, pseudogenes, ori's |
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| regulatory sequences |
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| coordinate gene expression - direct/regulate transcription |
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| mutant genes & fragments arise from… |
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| simple random mutagenesis or errors in DNA recombination |
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| reverse transcriptase |
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| • enzymes that copy RNA into dsDNA used by viruses • where pseudogenes come from |
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| miRNAs (microRNAs) |
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| • small structural RNAs (maybe >400 in human cells) • regulate expression of other genes by altering stability of product mRNA or ability to be translated |
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| repeated DNA |
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| • almost half of genome are repeats; two types: • microsatellite DNA • genome-wide repeats |
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| microsatellite DNA |
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| • very short, tandemly repeated sequences (<13bp; CACACA…) • from difficulties in accurately duplicated DNA • approx 3% of genome |
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| genome-wide repeats |
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| • much larger than microsatellite (>100bp, can be 1kb) • either as single copies throughout genome or clusters • all forms are transposable elements |
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| transposable elements |
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| • sequences that can "move" to diff places in genome • they multiply and accumulate throughout genome • rare process in human cells, but now 45% of genome |
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| transposition |
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| elements move to new position in genome, often leaving original copy behind |
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| important non-genetic portions of eukaryotic chromosomes |
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| • origins of replication: direct duplication of chrom DNA • centromeres: "handles" for movement of chromosomes into daughter cells • telomeres: protect and replicate ends of linear chrom |
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| origins of replication |
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| • sites where DNA replication machinery assembles to initiate replication • usually 30-40bp apart in eukaryotes • prokaryotes usually only have one ori |
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| centromeres |
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| • required for correct segregation of chromosomes after replication • they direct formation of elaborate protein complex, kinetochore • each chromosome only has ONE centromere |
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| kinetochore |
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| interacts w/ centromere DNA and microtubules (protein filaments) that pull sister chromosomes away from each other into two daughter cells |
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| telomeres |
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| located at the two ends of linear chromosome, bound by several proteins • proteins distinguish natural ends of chromosome from sites of chromosome breakage & other DNA breaks • act as specialized origin of replication - allows cell to replicate ends of chromosomes (recruits telomerase) • portion is in single-strand form and usually TG rich |
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| telomerase |
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| telomeres recruit this DNAP to faciliate end replication • during replication, lagging strand synthesized as short fragments (okazaki) --> RNAP removed by RNAse H, filled in by DNAPs, then ligated by DNA ligase • DNAP only able to add to 3' end - even if primase able to synthesize, DNAP cannot replicate DNA when primer removed = short region of unreplicated ssDNA left at end of chromosome • incomplete sections = end replication problem |
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| end replication problem |
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| • incomplete replication of 3' terminus of template DNA caused by exclusive 3' to 5' activity of DNAP • unreplicated ssDNA leads to loss of genes • ends of euk chromosomes called telomeres (GT rich repeats) - 3' end extends past 5' end • most euk use telomerase = protein + RNA (1.5 copies of compliment of telomeric sequence 3'-UAACCCUAA-5') • telomerase extends 3' end of telomere - does not need template; telomeric RNA serves as template • can be repeated many times, extending 3' end of chromosome • DNA replication machinery then extends 5' end of telomere • DNAP still cannot extend all the way to end of 5' • telomeric repeats protect by acting as buffer as non-coding DNA & proteins protects degregation of chromosomes |
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| chromosome structure changes |
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| ?after cell division occurs, chromosome structure altered many times; two states ® • interphase (chromosome decondensation) • M phase (chromosome condensation) |
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| SMC (structural maintenance of chromosome) proteins |
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| • form defined pairs by interacting through lengthy coiled-coil domains ® • cohesin & condensin • w/ non-SMC proteins, they form multiprotein complexes that link two DNA helices together • cohesion links sister chromatids together • condensin ring w/i individual chromosomes = tigher pack |
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| meiosis (2nd half of euk cell division) |
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| • specialized to produce cells w/ 1/2 # chrom than parent • follows DNA replication w/ 2 rounds of chrom segregation • Metaphase I, Anaphase I • Metaphase II, Anaphase II ® four gametes (or spores) • elongated G2 phase • monovalent attachment occurs in metaphase I • bivalent occurs in metaphase II |
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| meiosis I |
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| • homologous sister chromatids pair ® 4 chromosomes • chromatids from diff homologs recombine to form link btw homologous chroms ® chiasma • during metaphase I, two kinetochores from each pair attach to opposite poles (each monovalent) |
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| meiosis II |
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| like mitosis but instead of splitting chromatid pair into 2 cells, 2 sets of 4 chroms split into 4 cells ® dsDNA in each cell |
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| core DNA |
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| DNA btw each nucleosome ("beads on a string") |
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| linker DNA |
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| • DNA most tightly associated w/ nucleosome • typically only 20-60bp long (diff's from larger structures) |
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| nucleosome-free DNA |
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| typically associated w/ non-histone proteins for gene expression, replication, recombination |
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| histone-fold domain |
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| • mediates assembly of histone-only intermediates ; formation of head-to-tail heterodimers of specific histone pairs • w/o DNA, core histones form intermediate assemblies in solution • fold-domain is conserved region in each core histone, w/ 3 a-helical regions separated by 2 short, unstructured loops • H3 ; H4 form heterodimers, then together forms tetramer • H2A ; H2B stay as heterodimers |
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| histone tails |
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| • sites of extensive mods that alter fxn of indiv. nucleosomes • includes methylation, phosphorylation, acetylation • protease cleaves the tails, leaving histones intact |
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| dyad axis |
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| approximate twofold axis of symmetry in nucleosomes |
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| DNA Polymerization |
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| • DNAP is enzyme that catalyzes synthesis of new DNA • 3 Domains: Palm, Finger, Thumb |
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| DNA Synthesis |
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| • Two things needed for DNA synthesis ® • dNTPs (deoxynucleoside triphosphates) • primer:template junction • each of 4 dNTPs have 3 phosphoryl groups attached to 5' OH of 2' deoxyribose (named a, b, and g phosphates) |
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| primer:template junction |
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| • primer template junction has two components ® • template provides ssDNA to be copied • primer provides free 3' OH at growing end of DNA • 3'OH of primer attacks a-phosphoryl of incoming dNTP • leaving group is pyrophosphate w/ b and g • pyrophosphate (rapid) hydrolysis by pyrophosphatase provides additional free energy to drive reaction into two phosphate groups (b and g) • process can be repeated w/ 3' OH of new dNTP as nucleophile • chemistry requires that DNA be made in polar fashion (extending 3' end of primer strand, so 5' ® 3') • template strand directs what dNTP is added |
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| DNAP Domains |
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| • Palm: active site for DNA • correct bp important for catalysis reaction to continue • Also binds 2 divalent metal ions for DNA polym activity • once correct bp formed, finger encloses dNTP • conformational change brings dNTP and primer into correct orientation w/ metal ions |
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| Palm Domain |
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| • recognizes dNTPs vs rNTPs (even though rNTPs more) • it can sterically disclude rNTP's because w/ 2'OH, it's too small to fit |
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| Finger Domain |
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| • metal ion A deprotonate 3'OH of primer, producing oxyanion to attack a-phosphate of incoming dNTP • metal ion B coordinates (-) charges of b and g phosphates of dNTP ; stabilizes pyrophosphate leaving group • Finger residues also help to ® • Lys ; Arg stabilize pyrophosphate • Via stacking interactions, Tyr helps hold dNTP in place for catalysis • Finger Domain also associates w/ template, turns it 90o to avoid confusion of template vs. primer in palm's active site • only single template base in active site so palm knows what base to add to primer next |
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| Proofreading (in palm domain) |
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| • it hydrogen bonds to base pairs in minor groove of new DNA • h-bonds only form if nucleotides correctly base paired • if mismatched bp added, replication rate slows • primer template free to move around exonuclease site • exo site removes incorrect bp from 3' DNA end site backwards • primer:template slides back to DNA replication site |
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| Thumb Domain |
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| • no interaction w/ catalysis process • interacts w/ DNA most recently synthesized • reduces rate of dissociation btw junction ; DNAP • holds primer:template junction in active site |
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| DNA replication |
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| • Parental DNA copied to form two daughter DNA molecules • leading strand: pulled to right, copied continuously • lagging strand: copied backwards, discontinous, okazaki fragments; 100-1000bp in length |
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| Lagging strand |
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| • DNAP has to add to 3' end of lagging, opposite of leading • therefore, DNAP must move opposite direction, only adding discontinuously in small fragments • short fragments are okazaki fragments (100-1000bps) |
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| DNA helicases |
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| • Parental DNA must be copied into two daughter • enzymes that couple ATP hydrolysis to separation of DNA • hexameric proteins in shape of ring • junction btw new separated template strand & unreplicated dsDNA called replication fork • moves continuously through unreplicated dsDNA |
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| SSBs (single-stranded DNA binding proteins) |
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| • bind to ssDNA to stabilize separated strands |
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| topoisomerases |
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| • as DNA unwinds, twist number decreases • write number increases = (+) supercoiled DNA • Topo's remove (+) supercoils = (-) supercoil |
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| primase |
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| • specialized RNAP that makes short RNA primers using ssDNA as template • DNA primase activated by interacting w/ DNA helicase |
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| DNA polymerazes (DNAPs) |
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| • synthesis of DNA catalyzed by DNAP • can only add dNTPs to 3' OH of polynucleotide • bcz DNA antiparallel, one strand synthesized continuously towards repl fork, other is synthesized discontinously away from repl fork • takes 1 second for DNAP to bind to DNA • can add up to 1000 nucleotide bases per second |
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| processivity |
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| ability of an enzyme to catalyze reactions before releasing substrate |
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| sliding DNA clamps |
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| • binds to DNAP, holding DNAP & DNA together • surrounds DNA to increase processivity of DNAP |
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| RNAse H |
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| • To complete replication, RNA primers must be removed • RNAse H degrades RNA bp'd to DNA (H = hybrid for RNA:DNA) • single ribonucleotide directly linked to DNA removed by exonuclease (changes based on euk vs. prok) • single-strand gaps left behind by RNAse H are filled in by DNAP's |
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| DNA ligase |
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| • nicks btw 3'OH of repair section and 5' phos of replicated section repaired by DNA ligase |
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| Prokaryote Replication |
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| • two DNAP's to replicate leading/lagging strands often linked in holoenzymes • trombone model? ssDNA template on lagging pulls through DNAP, allowing DNAP to add nucleotides to 3' end of growing strand • sliding clamps act as loaders - help DNAP to find primed DNA |
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| Holoenzyme |
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| multi-protein complex where core enzyme activity (i.e. DNAP) associated w/ additional components that enhance function |
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| Semi-Conservative Replication |
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| in each new DNA double helix, one strand is from the original molecule, and one strand is new |
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| Where does prokaryotic replication take place? |
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| In the cytoplasm |
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| Where does eukaryotic replication take place? |
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| In the nucleus during S phase of the cell cycle |
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| What is the purpose of DNA replication? |
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| Duplicate chromosomes, so that after mitosis each daughter cell will inherit a complete genome |
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| In what type of cells does DNA replication occur? |
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| In dividing cells only; non-dividing cells are blocked in G0 and do not progress into S phase, so they do not replicate their DNA |
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| What are the requirements for DNA replication? |
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| DNA polymerase, Mg2+, template, primer, and dNTPs |
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| In what direction does DNA replication occur? |
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| 5` ® 3` direction |
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| What does complementarity mean? |
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| For each A on the template strand, there is a corresponding T added to the new strand. The same applies for a G on the template strand. A corresponding C is added to the new strand |
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| What enzyme is reponsible for removing mismatched nucleotides? |
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| 3`®5` exonuclease |
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| Bidirectional means? |
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| Replication proceeds in both directions from central origins of replication (ori) |
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| The type of replication in which the lagging strand is synthesized |
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| Discontinuous replication |
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| DNA in a newly synthesized daughter chromosome contains one new strand and one template strand. This is known as? |
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| Semiconservative replication |
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| DNA polymerase does what? |
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| It is an enzyme that catalyzes the polymerization of dNTPs into DNA |
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| How many types of DNA polymerases do prokaryotes have? |
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| I, II, and III |
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| What are the different types of DNA polymerases in eukaryotes? |
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| Pol-delta, pol-alpha, pol-beta, pol-gamma |
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| Pol-delta: location, function, processivity, proofreading, use of RNA primer |
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| • Location: nucleus; function: leading strand synthesis • processivity: >100,000 bp; proofread: yes; use RNA primer: yes |
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| Pol-alpha: location, function, processivity, proofreading, use of RNA primer |
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| • Location: nucleus; function: lagging strand synthesis • processivity: ~180 bp; proofreading: no; use RNA primer: yes |
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| Pol-beta: location, function, processivity, proofreading, use of RNA primer |
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| • Location: nucleus; function: fill in gaps for repair; • processivity: ~20 bp; proofreading: no; use RNA primer: no |
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| Pol-gamma: location, function, processivity, proofreading, use of RNA primer |
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| • Location: mitochondria; function: synthesis of both strands • processivity: ~8,300 bp; proofread: yes; use RNA primer: no |
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| The cofactor that is required for DNA polymerase activity? |
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| Mg2+ |
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| The pre-existing strand read by DNA polymerase is known as? |
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| The template strand |
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| What are the building blocks of DNA? |
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| dNTPs (deoxynucleotides) = dCTP, TTP, dGTP, dATP |
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| What is significant about dNTPs? |
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| They lack a 2` hydroxyl group |
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| How is thymine different from uridine? Where is uridine used? |
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| It is methylated at the 5` position; uridine is used for RNA |
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| Where does nucleotide polymerization get its energy from? How is this energy stored? |
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| Hydrolysis of triphosphate bonds; energy stored in triphos bonds as electrostatic repulsion of negatively charged O2s |
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| T/F: DNA polymerase can bind a 5` phosphate with an incoming 3` -OH? |
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| F; DNA polymerase can only bind a 3` -OH with a 5` phosphate of an incoming nucleotide |
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| What is a primer? |
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| It is a strand with a free 3` -OH group; DNA polymerase can only bind a 3` -OH with a 5` phosphate of an incoming nucleotide |
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| What direction does DNA synthesis proceed in? |
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| 5` ® 3` |
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| What direction is the template strand oriented? |
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| 3` ® 5`; it is antiparallel to the newly synthesizing strand |
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| Which DNA polymerases have the ability to proofread and determine if the new nucleotide is complementary to the corresponding base of the template? |
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| Pol-delta and pol-gamma |
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| What happens if an incorrect nucleotide is incorporated? |
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| The polymerase's 3`-5` exonuclease activity "kicks back", excising the mismatch; pol-alpha and pol-beta lack this activity |
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| The leading strand |
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| Continuously synthesized in the 5` - 3` direction |
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| The lagging strand |
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| • The opposite strand synthesized; • antiparallel to leading strand • discontinuously synthesized in 5`® 3` direction • stretches known as Okazaki fragments |
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| How long are Okazaki fragments? |
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| 100-200 bp |
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| What does semiconservative mean? |
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| It means that each chromatid receives one de nova and one parental strand; DNA replication is always semiconservative, never conservative nor non-conservative |
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| What is the origin of replication (ori)? |
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| It is the sequence where DNA replication begins |
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| How many ori's do prokaryotic chromosomes have? How many do eukaryotic chromosomes have? |
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| Prokaryotes = 1 ori per chromosome; eukaryotes = multiple ori per chromosome |
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| What is the general procedure of DNA replication? |
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| Factors recognize and bind specific ori sequence, unwind DNA, attract components of replication apparatus, apparatus moves down chromosome (away from ori), replicate DNA as it goes; DNA replication is bidirectional, two apparatuses assemble around ori |
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| What is a replicon? What is their average length? |
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| Region of eukaryotic chromosome that is replicated as unit, from one central ori; length about 200 kb (this is about the length of DNA loops anchored to scaffold proteins, suggesting these proteins may be involved in defining replicon length) |
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| Why is it a benefit to have multiple replicons? |
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| So that different regions of the genome can be replicated simultaneously; replication begins at the ori in the center of the replicon and extends in both directions until it reaches the end of an adjacent replicon |
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| What is the purpose of helicase? |
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| Enzyme that unwinds DNA; one of the first factors to bind ori; serves to open double helix so DNA polymerase can replicate strands; is part of replication apparatus |
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| What are single stranded bindng proteins (SSB)? |
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| Factors that stabilize single stranded DNA by preventing it from winding back into double helix |
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| What does the DNA replication apparatus consist of? |
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| Helicase, DNA polymerase-delta, pol-alpha, beta-clamp, and primase; two apparatuses assemble, one on each side of the ori, and each moves in the oppostie directions, replicating DNA as they go |
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| What does the beta-clamp do? |
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| Ring-like protein that wraps around DNA to stabilize association of replication apparatus; required for pol-delta processivity; without clamp = pol-delta only replicates short oligonucleotides (< 200 bp), with clamp = stretches > 100 kb are produced |
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| Strand that is continuously synthesized, in the 5`-3` direction? |
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| Leading strand |
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| What is DNA polymerase-delta? |
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| Enzyme that replicates leading strand, reads template one base at a time, incorporates complementary nucleotides and ligates their 5` phosphate to the 3` -OH of the growing strand |
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| What is the overall synthesis of the lagging strand? How is its synthesis overcome? |
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| Overall synthesis is in the 3`-5` direction (wrong direction); its synthesis is overcome by synthesizing discontinuous short stretches called Okazaki fragments |
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| What does primase do? |
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| Enzyme that binds unwound lagging strand and transcribes short stretches of RNA (< 15 bp); RNA serves as primer, providing 3` -OH group required by pol-alpha |
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| What is the purpose of DNA polymerase-alpha? |
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| Enzyme uses RNA primer to synthesize Okazaki fragments of the lagging strand; NO proofreading ability; only synthesizes one fragment at a time while helicase contiues to unwind DNA for next fragment, so lagging strand held in loop |
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| What happens when pol-alpha reaches a primer at the end of a previous fragment? |
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| Lagging strand is released, primase makes next primer at end of new single stranded region, process is repeated |
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| What are the three enzymes involved with removal of RNA primers from the lagging strand? |
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| RNAase, DNA polymerase-beta, DNA ligase |
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| What does RNAase do? |
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| This enzyme digests any RNA; in replication it serves to remove lagging strand primers |
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| What does DNA polymerase-beta do? |
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| It fills in the gaps in DNA (~ 20 bp); in replication it fills in the gaps left after RNA primers are removed; N.B. = DNA polymerases can only add nucleotides, they cannot link DNA fragments (pol-beta leaves nicks in DNA) |
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| What does DNA ligase do? |
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| Enzyme that binds any free 3` -OH and 5` phosphates of DNA; in replication it seals the nicks between Okazaki fragments left by pol-beta |
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| What happens as a result of helicase unwinding? What enzyme helps fix the problem? |
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| Supercoiling increases; topoisomerases helps to restore DNA to its proper level of supercoiling |
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| What happens when the last RNA primer is removed from the end of the chromosome? |
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| It leaves an overhang that cannot be filled by DNA polymerase; if this didn't get resolved, then every time dividing cells replicated their DNA, the lagging strands would lose a bit of their telomeric sequence, leading to chromosomal destabilization |
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| What enzyme is responsible for filling in the gap left when the last RNA primer is removed? |
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| Telomerase (it is a type of reverse transcriptase) - it fills in the gaps as well as extends the length of the telomere |
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| What are some interesting facts about telomerase? |
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| Activity and length decreases with age; primary cell culture lines have no telomerase activity; immortalized cell culture lines divide indefinitely b/c of activated telomerase; activated in cancer cells and believed to contribute to immortalization |
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| Where is gene expression controlled? |
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| Pre-transcriptional, transcriptional, post-transcriptional, translational, and post-translational levels |
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| DNA replication must be these three thing |
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| • high fidelity (but need mutations) • highly processive • relatively fast |
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| Semiconservative replication |
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| • both strands get a new stand • this is the correct method |
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| conservative replication |
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| • both strands stay connected and a new one is created |
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| dispersive replication |
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| • random pieces are kept and others are replaced |
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| Meselson and Stahl experiment |
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| • demonstradted semiconservative • applied CsCl gradient ultracenterfugation of DNA labeled with Heavy Nitrogen (15N) • cells moved to growth medium containing normal N (14N) • DNA was isolated at different times • pictures taken during centerfugation • found old strand of heavy DNA with new strand of light DNA |
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| continuous replication |
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| both strands serve as templates in the same compass direction one would do 3'®5' and hte other would do 5'®3' |
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| semidiscontinuous replication |
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| same as continuous, but broken up into bursts on the same double strand |
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| discontinous replication |
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| fragmented on both parent strands |
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| Okazaki proposed |
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| • proposed that DNA polymerase can make one strand of DNA continously 5'®3' (leading strand) • but the other strand would be discontinous in 5'®3' (lagging strand) |
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| Okazaki's model had two experimentally testable predictions |
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| • if short pieces of DNA are synthesized on lagging strand, should be able to catch w/ radiolabeling • if the enzyme DNA ligase is eliminated from the replication process, then short pieces of DNA made should be detectable even at longer labeling periods |
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| Okazaki's experiment |
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| • used T4 phage mutant that made Ligase • control - found short pieces for short periods of time before they were ligased together • using the T4 mutant - tons of short pieces as time increased |
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| DNA replication is _____ with DNA synthesis occuring ____ at ____ _____ ____ |
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| • bidirectional • simultaneously • 2 replication forks |
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| specific location where replication starts |
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| • oriC • contains 4 9-mers having consensus sequence of TTATCCACA |
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| helicase |
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| • an ATP-dependent enzyme that separates the DNA strands in advance of the replication fork • the dnaB gene product in E.coli |
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| Single-Stranded DNA-Binding Proteins |
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| • bind to ssDNA and prevent it from reforming dsDNA • product of ssb gene of E.coli • the bonding is cooperative - raises affinity by 1,000 fold for next molecule • stimulate replication |
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| Topoisomerase |
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| • as the DNA unwinds, it has to wind somewhere upstream, creates strain • topoisomerase releaves this strain by introducing temporary single- (Type I) or double-stranded (Type II) breaks in the DNA |
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| DNA gyrase |
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| • from E. coli • type II topoisomerase |
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| DNA Polymerases found in E. coli |
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| • Polymerase I - DNA repair, primer excision • Polymerase II - SOS repair • Polymerase III - required for DNA replication in E.coli |
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| Holoenzyme polymerase III subunits (aka: pol III holoenzyme) |
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| • composed of multiple subunits • The ?-subunit has the DNA polymerase activity • The ?-subunit has the 3’ a 5’ exonuclease proofreading activity • The ?-subunit has the ???? activity. The function is still unknown |
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| eukaryotic DNA polymerases |
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| • DNA polymerase ? (priming) • DNA polymerase ? (elongation) • DNA polymerase ? (repair) • DNA polymerase ? (repair) • DNA polymerase ? (mitochondrial) |
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| DNA replication can be divided into 3 major events |
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| • initiation ® elongation ® termination |
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| initation of replication - purpose of dnaA |
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| • binds at the oriC • facilitates the binding of dnaB • stimulates melting of the 3 13-mer repeats at one end of the oriC to make an opening |
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| initiation of replication - purpose of dnaB |
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| • required for protein synthesis • dnaC binds to dnaB • stimulates binding of the primase • also serves as the helicase that moves 5'®3' on the lagging strand in the direction of the replication fork |
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| initiation of replication - two other factors for open complex formation at oriC |
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| • RNA polymerase which synthesizes a short piece of RNA that creates an R loop • helix unwinding (HU) protein which induces bending |
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| Initiation of replication - primase |
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| • product of the dnaG • its the RNA primer-synthesizing enzyme |
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| initiation of replication - Primase (dnaG) + dnaB = |
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| • primosome • this is responsible for laying down multiple primers for Okazaki fragments on the lagging strand |
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| how eukaryotes handle initiation |
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| • multiple sites of replication for each chromosome |
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| elongation - why pol III holoenzyme is highly processive |
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| • due to the sliding clamp • its the ?-subunit of the holoenzyme • literally holds the entire pol III assembly on the template for long periods |
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| elongation - group of protein required for the sliding clamp |
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| • the ?-complex • sliding clamp cannot touch the DNA by itself • serves as the clamp loader and is ATP-dependent |
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| elongation - eukaryotes version of the sliding clamp |
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| • processivity factor PCNA • PCNA = proliferating cell nuclear antigen • forms a trimer (3 subunits) that can encircle the DNA as the bacterial clamp does |
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| elongation - pol III holoenzyme |
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| • contains two core polymerases - one for each strand • as it finishes one Okazaki fragment, it runs into a nick that is positioned in front of the primer on the next fragment • this nick is a cue for the complex to dissociate from the and move to the primer on the next fragment |
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| termination - the _____ region |
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| • terminus • where the replication forks begin to get near each other • contains 22bp sites that bind specific proteins called TUS proteins • replication forks stop moving when they get to this region • leaves the daughter couples entangled |
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| TUS proteins |
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| • terminus utilization substance |
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| termination - when circular DNAs are interlocked (entangled), the structure is called ________ |
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| • a catenate |
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| termination - Decatenation |
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| • process of untangling the interlocking DNA rings • performed by topoisomerase IV |
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| eukaryotes' termination problems |
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| • there are gaps left when RNA primers are removed • this is problematic because when the primer is removed DNA cannot be extended in the 3'-->5' direction • and there's no 3' upstream • so DNA should be getting shorter… |
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| terminaton ends - which is which |
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| • TTGGGG - Tetrahymena • TTAGGG - vertebrates |
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| Hayflick Limit |
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| • Hayflick (1960s) showed that normal animal cell lines are not immortal • grow in cultures of about 50 generations, then enter senescence • cancer cells do not have this limit (contain telomerase as do egg and sperm production cells) |
