Chromatin Chromosomes Genome Lecture 3 Chromosomal DNA genome total gene c composi on of organism DNA in chromosomes long millions of bps humans have 1 000 fold more DNA than an E coli chroma n vs chromosomes chroma1n uncondensed gene c material condense gene c material into chromosomes to divide up during mitosis genome DNA long chroma n uncondensed chromosomes condensed Chromosomes How to Ensure Daughters Receive Full Set of Chromosomes 1 DNA replica1on origins ORI only 1 in bacteria but many in euks 2 centromere contains centromeric DNA special DNA only located centromere forms kinetochore makes MTs 3 telomeres special structures ends of chromosomes must protect chromosome ends if they re vulnerable get manipulated then cell will either arrest or undergo programmed cell death DNA replica on ORIs centromere centromeric DNA telomeres protect chromosome ends Chromosomes centromere primary constric on on chromosome required for mito c segrega on centromeric DNA forms centromere 1 2 3 CENPDA centromere protein histone H3 variant binds CBF3 complex CBF3 complex recruits Ndc80 Dam1 which ini ally bind MT s side but nish as end aTachment on MT Form mito c spindle from these kinetochore proteins Ndc80 Dam1 in order to sa sfy spindle checkpoint to leave metaphase start anaphase ALL sister chromosomes MUST be aTached to opposite spindle poles be under tension i e must be lined up a ached non disjunc on incorrect amount of DNA in cells occurs if metaphase happens when kinetochores aren t aTached to chromosomes CENTROMERE centromere constric on required for segrega on forming centromere CENPDA binds CBF3 CBF3 recruits Ndc80 Dam1 mito c spindle forma on to sa sfy spindle checkpoint start anaphase tension aTached to opposite poles centromere constrictor for segrega on forming centromere spindle checkpoint Chromosomes Centromere TELOMERES Structure telomeres protein DNA complexes ends of linear chromosomes G rich TTAGGG dsDrepeats bound by TRF1 TRF2 ss DNA bound by Pot1 telomerase telomeres protein DNA complexes ends of linear chromosomes containing telomeric sequences G rich TTAGGG double stranded dsDrepeats ds repeats bound by TRF1 TRF2 Telomere Repeat Factor other proteins recruited single stranded DNA ss DNA 3 overhang bound by Pot1 Protec on Of Telomere telomerase involved in replica on of telomere see DNA replica on lectures telomeres protein DNA complexes ends TTAGGG ds repeats TRF1 TRF2 ss DNA Pot1 Chromosomes Telomeres Structure TELOMERES TDLoop TDloop structure end of mammalian chromosomes ds DNA bound by TRF1 DDloop formed by 3 5 3 overhang bound by TDloop DDloop structure that protects telomere TRF2 ss DNA Pot1 T loop ds DNA TRF1 TRF2 D loop ss DNA 3 overhang Pot1 Chromosomes Telomeres Structure enable replica on of chromosome TELOMERES Func1ons cap protect chromosome ends ends Shelterin long telomeres associated w rapid end replica on cap protect ends long associated w rapid loss signals DNA damage cell growth response cell growth response loss of telomere signals DNA damage unprotected telomere ATM kinase p53 apoptosis OR senescence replicate ends cap protect long rapid cell growth loss signal DNA damage response Chromosomes Telomeres Func ons DNA Packaging Solving the Problem DNA Compac1on nucleosomes beadsBonBaB string 30Dnm ber beads wound up looped into solenoids Must Package Mammalian Nuclei DNA humans have 24 chromosomes each chromosome 50 250 million bps of DNA can be up to 8 5 cm long problem cell nucleus diameter only a few micrometers chromosomes are too long solu on DNA compac on nucleosomes 30 nm ber DNA Packaging Chroma n Chromosomes chroma1n complex of DNA proteins histones non histone proteins chromosomal proteins histones HISTONES Chromosomal Proteins chroma1n DNA proteins histones 1 1 ra o Lys Arg nucleosomal or non nucleosomal present in 1 1 ra o weight weight w DNA basic have high Lys Arg content histones have charge while DNA has charge histones DNA s ck together may be nucleosomal or non nucleosomal recruit factors that take away some of histones charge loosens DNA histone binding RNA pol can bind DNA interact w it chroma n histones 1 1 ra o w DNA lots of Lys Arg nucleosomal vs non nucleosomal Chromosomes Histones HISTONES Nucleosomal vs NonD Nucleosomal Histones nucleosomal histones small H2A H2B H3 H4 histone CORE nonDnucleosomal histones H1 larger Nucleosomal Histones small proteins H2A H2B H3 H4 form octamer form histone CORE DNA in nucleosome wraps around histone core NonDNucleosomal Histone H1 NOT in octamer larger than nucleosomal histones nucleosomal H2A H2B H3 H4 histone core non nucleosomal H1 Chromosomes Histones Nucleosomal Non Nucleosomal NUCLEOSOMES nucleosome chroma n packaging unit histone core DNA electron micrographs beads on a string nucleosome packaging unit of chroma n consists of histone CORE DNA revealed in electron micrographs as beads on a string afer treatment that disrupted higher order packing chroma n packaging unit histone core DNA beads on a string Chromosomes Nucleosomes NUCLEOSOMES Obtaining BeadsDonDaDString Water Model 1 Place isolated nuclei in water 2 Centrifuge on to carbon grids 3 Stain w uranyl acetate 4 Perform EM Trypsin Model 1 Digest chroma n w trypsin a protease to remove lysineBrich histones H1 or H5 2 Stain w uranyl acetate 3 Perform EM Micrococcal Nuclease MNse Caused String to Separate from Beads DNAse cuts DNA into pieces string disappeared afer nuclease MNse diges on mul mers of nucleosomes can be isolated on a sucrose gradient afer par al chroma n diges on w MNse releases 1 nucleosome core par cle water model trypsin model MNse nuclease sucrose gradient Chromosomes Nucleosomes Beads on a String Methods NUCLEOSOMES Separa1on of Components EM of Gradient Frac1ons 1 nucleosome 2 nucleosomes 3 nucleosomes 4 nucleosomes dissociate released nucleosome core par cle w high salt histone octamer dissocia on Chromosomes Nucleosomes disc shaped par cle diameter 11 nm contains 2 molecules each of H2A H2B HISTONE OCTAMER disc shaped 2 each of H2A H2B H3 H4 every 200 bps linker DNA separates octamers occur every 200 bps of DNA in chroma n linker DNA separates octamers from H3 H4 each other DNA winds around histone CORE 2x 83 bps per turn 83 x 2 166 bps some linker DNA lef over 200 bps disc shaped every 200 bps linker DNA Chromosomes Histones Histone Octamer beads on a string organized into 30DNM FIBER Higher Order Structure of Nucleosomes beads on a string organized into 30 nm bers histone tails H1 promote forma on