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Molecular Week 3 Notes

by: Kiara Lynch

Molecular Week 3 Notes Bio 413

Marketplace > La Salle University > Biology > Bio 413 > Molecular Week 3 Notes
Kiara Lynch
La Salle
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About this Document

These notes cover information from week 3: DNA replication
Dr. Stefan Samulewicz
Class Notes




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This 7 page Class Notes was uploaded by Kiara Lynch on Thursday February 11, 2016. The Class Notes belongs to Bio 413 at La Salle University taught by Dr. Stefan Samulewicz in Spring 2016. Since its upload, it has received 53 views. For similar materials see Molecular in Biology at La Salle University.


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Date Created: 02/11/16
Molecular Week 3 Notes  Chromatin Remodeling Complexes o Large protein complexes o Grabs onto linker region  Wedges itself between DNA and nucleosome o Induces conformational changes o Pries apart nucleosomes o Dissociates region of DNA from packaging  exposes gene o Nucleosome sliding catalysis by ATP dependent chromatin remodeling complexes  Pushes on DNA and loosens attachment to nucleosome core  Each cycle of ATP binding and hydrolysis and release of ADP + P moves DNA with respect to the histone octamer o Nucleosome removal and histone exchange or shifting of core nucleosome proteins  Remove H2A and H2B dimers and replace with dimers that contain a variant histone  Remove octamer completely and/or replace with a different nucleosome core o Tail- post translational modifications to amino acid side chains in tails  Use tails as a code/signal that determines how densely packed that segment gets; 1 or 2 modifications per tail; 4 core histone tails  Lysine acetylation and methylation are competing reactions  Makes hydrophilic or hydrophobic  Serine Phosphorylation  Reader proteins- have specific binding sites; wraps around tail and binds to post-translational modifications  Code reader complex o Scaffold binds reader proteins and brings together and interprets what it is seeing and attracts other components conformational changes in scaffold protein occur binding of other proteins (ex: chromatin remodeling complex) o Binds only to region of chromatin that contains several of the different histone marks that it recognizes o Only specific combinations of marks will cause binding and attraction of other proteins  Ex: protein molecule specifically recognizes Histone 3 (H3); N-terminal 6 amino acids are recognized; PHD domains recognize methylated lysines on histones  Tail modifications for gene silencing, gene expression, gene inactivation, etc. CHAPTER 5  Mutations o Occur randomly to DNA o Examples  Fibrinopeptides- blood clots when these peptides are cut; negative charges repel each other and keep soluble  Hemoglobin- delivers oxygen to the body; tetramer  Cytochrome C o Sexually reproducing organisms  Germ line cells propagate genetic info to the next generation  Somatic cells are necessary for survival of the germ line cells but do not have progeny  DNA replication o Split two strands o Need energy source, building blocks o Correct base pairing o Need to keep two strands apart; Need to transcribe in correct direction o Need proofreading and ability to repeat o Semiconservative nature of DNA replication- original strands remain in tact 3  H -Thymidine Experiment- Pulse-chase o Looking for something you can’t see ordinarily  formation of DNA o Label DNA in a sensitive way  Radioactive isotope triduum H3  Incorporate into building blocks of DNA o Trying to get a snapshot of a moving process o Add for certain period of time (30s)  Stop or add chase  Flood with nonradioactive thymidine and let process continue  Pulse, no chase; pulse 30s, chase 30s; pulse 30 s, chase 1 min  Shows length of DNA increasing  Replication fork structure o Lagging strand- pulls apart  Made up of Okazaki fragments o Leading strand o 5’ to 3’ synthesis  Laggings strand must be made initially as a series of short DNA molecules (Okazaki fragments)  Synthesized sequentially; those nearest to the fork are the most recent  DNA replication o Necessities  Template strand  Building blocks GATC  Free 3’ hydroxyl o Separate 2 strands using DNA helicase  Binds single stranded DNA  Wraps around single strand  Uses ATP; hydrolysis causes conformational changes  Propels along single strand and separates the double strand o Need strands to stay apart long enough to be synthesized  Single strand binding proteins- proteins with domains that bind to backbone of single stranded DNA- cooperative protein binding straightens region of chain  Hairpin loops- looks like double stranded DNA so DNA polymerase stops o Each domain has binding sites- no interaction with bases; sequence is independent  Keeps DNA single stranded o DNA polymerase  DNA dependent DNA polymerase  Catalyzes stepwise addition of DNA to 3’ end  primer strand  Nucleotide in  conformational change with ATP hydrolysis  active site opens up and bumps 1 nucleotide over o DNA primase  DNA dependent RNA polymerase  Enzyme to produce part of template to give 3’ hydroxyl  10-12 RNAs  Can start a new polynucleotide chain by joining 2 nucleoside triphosphates together  5’ to 3’ then stops  makes 3’ end available for DNA polymerase o Synthesis on lagging strand  RNA primers made at intervals  Primers erased by DAN repair enzyme (RNase H) that recognizes RNA in RNA/DNA helix and fragments it  creates gaps  gaps filled by DNA polymerase and ligase  DNA ligase links okazaki fragments o DNA ligase reaction  Prime 5’ end break bond between 2 phosphates link 5’ and 3’ ends  Enzyme seals a broken phosphodiester bond  Uses ATP to activate 5’ end at the nick and forms a new bond  Energetically unfavorable nick-sealing reaction is driven by being coupled to the energetically favorable process of ATP hydrolysis o Clamp protein  Want polymerase to hold on tightly until it gets to the end of a strand  Core of alpha helices surrounding DNA strands; Beta sheets on outside  Holds polymerase on while it makes a new strand  Holds tight when polymerase is moving and lets go when it stops o Clamp loader protein  Attracts clamp protein  Matches grooves of DNA  Tightens around primer junction until its further progress is blocked by 3’ end of primer  loader hydrolyzes ATP and releases clamp  Clamp loader dissociates into solution once clamp is assembled  At a true fork, the clamp loader remains close to lagging strand ready to assemble another clamp at each okazaki fragment  Wraps clamp around DNA  DNA attracts polymerase  ATP hydrolysis  clamp holds onto polymerase  clamp loader releases o Replication fork  Helicase- 1000 nuc/sec  Lagging strand- folded to bring polymerase into a complex with leading strand  Folding brings 3’ end of okazaki fragments close to start site of next fragment o Mistakes  Mismatch due to incorporation of a rare, transient tautomeric form of C  Temporarily looks like a T  shift back to C but can no longer base pair with A  free 3’ hydroxyl flopping around o If hydroxyl is not in the right place the process stops  Part of DNA polymerase that removes the misincoporated nucleotide is an exonuclease that cleaves nucleotides 1 at a time from the ends of the polynucleotide  DNA polymerase  Chews backward until it finds a properly base paired segment  3’ to 5’ exonuclease  Prevents point mutations o If it is the wrong nucleotide the active site (“hand”) does not close o The DNA is moved to another active site  editing site  AT GC proofreading; site directed mismatch repair  Growth in 3’ direction allows chain to continue to be elongated when a mistake in polymerization has been removed by exonuleolytic proofreading  5’ direction blocks chain elongation- active site is on the wrong side  Strand directed mismatch repair  MutS identifies and binds to mismatched base pairs and MutL scans nearby DNA for a nick and decides which strand to remove (mismatch usually in newly formed strand)  Triggers degredation of nicked string all the way back through the mismatch  Winding problem  Knots form ahead of replication fork  Must rotate to prevent knots (polymerase moving at 500 nucleotides/second and unwinding of helix ahead of fork at 50 revolutions/second)  Topoisomerases o Ahead of replication fork o Remove stress due to rapid unwinding o Tyrosine binds and breaks backbone in 2 strand  frees up other strand to spin around phosphodiester bond  unravels in opposite direction  reattaches backbone o Catalyzed by eukaryotic DNA topoisomerase I enzyme- form single covalent bond with DNA which allows free rotation around covalent backbone o Origin of Replication  Replication bubble formed by replication fork initiation  Both strands are separated and serving as templates for DNA synthesis  Pulse-chase  Showed direction of replication bidirectional elongation (2 forks)  Initiator Proteins- prokaryotes  Identify sequence in genome and binds  Recruits helicase  Helicase temporarily inhibited by binding (initiator) protein  Helicase unwinds DNA  Binds to specific DNA sequences in replication origin and forms a compact structure  DNa wraps around protein  helicase/loading protein inhibited until at replication origin (prevents from inappropriately entering single stranded DNA  opens DNA  primase synthesizes primer  initiation of 3 DNA chains and assembly of 2 replication forks  Identifying sequences sufficient for initiating DNA replication- eukaryotes o Experiment: randomly selected yeast DNA segment and segment of yeast DNA containing ARS (gene responsible for Histone synthesis); introduce plasmid DNA into yeast cells  Yeast cells that lack HIS gene cannot grow in absence of Histidine  Grow in medium without histidine  Transformation in cells with ARS; replicate free of host chromosome o Cut up genome  ligate pieces into plasmids  Plasmid- vehicle used to move chunks of DNA around  Characteriscs that allow it to function as a natural piece of DNA (get passed down like chromosomes)  About 150 bp long  Binding sites  Stretch of DNA easy to unwind (AT rich- only 2 H bonds so easy to break apart)  Abf1 helps attract origin of replication complex o DNA replication in eukaryotes  New complex cannot form at origin until cell progresses to next G phase- makes sure it fires only once  2 proteins that bind to ORC and attract helicase- Cdc6 and Cdt1  Phosphorylate ORC- don’t repeat replication  Before synthesis phase  3.2 million nucleotides to make DNA  Package DNA with nucleosomes- synthesize proteins o Distribution of parental and newly synthesized histones behind replication fork  Random distribution- roughly equal numbers inherited by each daughter see  Most are hybrids  Chaperones restore full complement of histones to daughter molecules  H2A- H2B dimers that are out of nucleosomes need to be replaced; dissociate more readily  Chaperone proteins- take dissociated or newly synthesized core histones and bring to daughter DNA to reconstitute o Parental patterns of histone H3 and H4 modification can be inherited by daughter chromosomes  Reader writer complexes-read modification and write similar modification onto adjacent nucleosomes  Modifications to histone tails on both strands o Telomerase- protein-RNA complex  RNA dependent DNA polymerase  Remainder of telomerase RNA acts as a template that will be copied to make telomere  Telomeres require telomerase  Synthesis by reverse transcriptase  Polymerase enzyme that uses RNA template to make DNA strand  Carries its own RNA template  At ends of chromosomes  Synthesize repeating G rich sequences at ends  Add nucleotides until appropriate length  Always a little bit of single stranded DNA that forms a T loop  T loop- decides optimal length for telomere; put in extra- long and extra short ones and saw what happened over time cells corrected the length  Repeat sequences; telomerase copies repeat sequence of template and keeps chromosomes from shortening  3’ end extended by RNA-templated DNA synthesis  Lagging strand- completed by DNA polymerase alpha (has subunit of DNA primase)


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