Chromosomes and DNA Packaging--lecture

8/7/2019 Chromosomes and DNA Packaging--lecture

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Chromosomes andChromosomes and

DNA PackagingDNA Packaging

Chapter 5Chapter 5


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T he ProblemT he ProblemHuman genome (in diploid cells) = 6 xHuman genome (in diploid cells) = 6 x1010 99 bpbp6 x 106 x 10 99 bp X 0.34 nm/bp = 2.04 x 10bp X 0.34 nm/bp = 2.04 x 10 99 nmnm= 2 m/cell= 2 m/cellVery thin (2.0 nm), eVery thin (2.0 nm), extremely fragilextremely fragileDiameter of nucleus = 5Diameter of nucleus = 5- -1010 QQmm@@ DNA must be packaged to protect it,DNA must be packaged to protect it,but must still be accessible to allow genebut must still be accessible to allow geneexpression and cellular responsivenessexpression and cellular responsiveness


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S olution: ChromosomesS olution: ChromosomesS ingle DNAS ingle DNAMolecule andMolecule andassociatedassociated

proteinsproteinsKaryotypeKaryotypeChromatin vs.Chromatin vs.

ChromosomesChromosomes


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HISTO NESHISTO NES

Main packaging proteinsMain packaging proteins

5 classes: H1, H2A, H2B, H3, H4.5 classes: H1, H2A, H2B, H3, H4.Rich in Lysine and ArginineRich in Lysine and Arginine


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HISTO NES HISTO NES NOT E: if histones from different speciesNOT E: if histones from different speciesare added to any eukaryotic DNA sample,are added to any eukaryotic DNA sample,chromatin is reconstituted. Implication?chromatin is reconstituted. Implication?Very highly conserved in eukaryotes inVery highly conserved in eukaryotes inbothboth

S tructureS tructure

FunctionFunctionProtectionProtectionMust allow gene activityMust allow gene activity


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Variability in HistonesVariability in Histones


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HISTO NESHISTO NES

Fairly uniform,Fairly uniform,some variabilitysome variabilityWhy variable if onlyWhy variable if onlyfunction isfunction ispackaging?packaging?


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Variations In HistonesVariations In HistonesHow can cells introduce changes in proteinHow can cells introduce changes in proteinstructure and thus protein function?structure and thus protein function?

MutationsMutationsPost transcriptional modificationsPost transcriptional modifications ex alternateex alternatesplicingsplicingPost translational modificationsPost translational modifications

AcetylationAcetylationMethylationMethylationS

erS

er--T

hrOT

hrO

--phosphorylationphosphorylationHis NHis N--phosphorylationphosphorylation

NOT E: These processes are dynamic. They give theNOT E: These processes are dynamic. T hey give thecell another means to regulate gene expressioncell another means to regulate gene expression


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O rders of chromatinstructure fromnaked DNA tochromatin to fully

condensedchromosomes...

Fig. 9


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Beads on a S tringBeads on a S tring 10 nm Fiber10 nm Fiber


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10 nm filament; nucleosomes

histones(= 1 g per g DNA) DNA

proteinpurification

H 1

H3H2A

H2B

H4

Basic (arg, lys);

+ charges bindto - phosphateson DNA


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deoxyribonuclease I(DNase I) digestion

nucleosomes

Conclude: histones in anucleosome protect 1 46 ntfrom DNase I attack.

Separate DNA

from protein

H 1

2H32H2A

2H2B2H4

the histoneoctamer

proteins1 46 nt fragments

DNA


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10 nm Fiber10 nm Fiber

A string of nucleosomes is seen under EM as a 10 nmA string of nucleosomes is seen under EM as a 10 nm

fiberfiber


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30 nm Fiber30 nm Fiber

30 nm fiber is coil of 30 nm fiber is coil of nucleosomes with 6/turnnucleosomes with 6/turn


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T he 30 nm FiberT he 30 nm Fiber(Compacts DNA 7X more)(Compacts DNA 7X more)


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O rders of chromatinstructure fromnaked DNA tochromatin to fully

condensedchromosomes...

Fig. 9


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Acid extraction removes histones from meta-phase chromosomes, leaving nonhistones ...

Low power high power

Scaffoldprotein

DNAloops

Major non-histone proteins = topoisomerases!


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In sperm heads, DNA must be furtherIn sperm heads, DNA must be furthercompressedcompressedProtaminesProtamines~2/3~2/3 rdrd basic amino acids, (mostly Arg)basic amino acids, (mostly Arg)Greater degree of compaction associatedGreater degree of compaction associatedwith:with:

Fit in DNAFit in DNAS alt bridges between Arg residues in both theS alt bridges between Arg residues in both thecontact helix and adjacent helicescontact helix and adjacent helices

ProtaminesProtamines


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O ther DNA Associated ProteinsO ther DNA Associated Proteins

Gene expression in a dynamicGene expression in a dynamicenvironment environment DNA associated nonDNA associated non- -histones:histones:

PalindromesPalindromesEE --heliceshelicesS ymmetrical dimersS ymmetrical dimers

Interaction between RInteraction between R- -groups and specificgroups and specificbasesbasesRole of basic amino acidsRole of basic amino acids


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backback


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Phosphorylation ( S er and T hr)Phosphorylation ( S er and T hr)backback


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Histidine: NHistidine: N--

phosphorylationphosphorylationHow will this affect How will this affect His s ability toHis s ability toparticipate in weakparticipate in weakinteractions?interactions?

BackBack


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E uchromatin (E ) vs Heterochromatin (H)

EH

DNase

Being more condensed (tightly packed),heterochromatin is resistant to DNasedigestion.

Fig. 11


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Hypothesis:Transcriptionally active DNA (i.e., an

active gene) is in euchromatin.

Can test with DNase I, which shouldpreferentially digest active genes in

a cell...

E

H

nascent transcripts


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An E xperiment:chick red blood cells chick RBC nuclei

chromatin extractiontotal chromatin

digest with DNase I for different times

partially digestedchromatin; extractundigested DNA

denature DNA, spot on filter

Increasing time of digestion

make 2of these...

Fig. 12


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Radiolabel, denature cloned globin and other geneDNA to use as *probe .

Allow * probe DNA to hybridize to complementarysequences on filters e xpose, develop autoradio-graph...

globin gene probe

ovalbumin geneprobe

Fig. 13

Increasing time of digestion

DNA from digested RBC chromatin


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Compared to globin gene DNA, ovalbumin geneDNA in RBC chromatin resists DNase digestion.

Globin gene DNA in RBC chromatin must bemore accessible to DNase (less condensed,more euchromatic) than ovalbumin DNA.

Conclusions:


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HistonesHistones Degrees of ConservationDegrees of ConservationH4H4------ O nly 2 variations ever discoveredO nly 2 variations ever discoveredH3H3 also highly conservedalso highly conservedH2A, H2BH2A, H2B------ S ome variation betweenS ome variation betweentissues and speciestissues and speciesH1H1--like histoneslike histones

H1H1 Varies markedly between tissues andVaries markedly between tissues and

speciesspeciesH1H1 ----Variable, mostly present in nonVariable, mostly present in non- -replicating cellsreplicating cellsH5H5------Extremely variableExtremely variable


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Post Post--T ranslational ModificationsT ranslational Modifications


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30 nm solenoid fiber10 nm filament;nucleosomes

[salt]

Dissection of interphase nuclei...

low [salt] extraction

Chromosomes and DNA Packaging--lecture

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