Chromosomes and Replication

8/9/2019 Chromosomes and Replication

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Vandan DesaiBIOL 303Cell Biology

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CHROMOSOMES and REPLICATIO (Study Guide)

I. ReviewA. Basic functions of chromosomes (CH):

i. (Package) Package DNA intosmaller volume DA = 6ft long & Cell= 10 20 m

ii. (Distribute) Help to ensure equal segregation of each CH at mitosis Minimize tanglingand traffic jams Ensure that each cell gets 23 types of CHs

iii. (Express) Help to regulate gene expression Humans have ~30,000 genes but not all active simultaneously (so, want

to be able to turn on/off when want to)B. Terminology:

i. Chromosomegenetic linkage group w/ one piece of DNA All genes on one CH linked together Structure ofmaximally condensedDNA + protein = mitosis (metaphase) Parts:

a. Telomere = endsb. Kinetochore = spindle fiberattachment site; at centromere

i. Microtubules that are at site forseparation of chromatidsii. Protein structure w/ repetitive DA

Duplicated CH at metaphase has twochromatidsa. Exist after DA replication and till anaphase (called chromatids)b. During anaphase, each become chromosome when move toward

poles (called chromosomes)ii. Chromatinunwound, uncondensedchromosomes; exist in non-mitotic cells

eu- = more/most unwound; contain active genes; CH go thru mitosis hetero- = compacted; little/not unwound; inactive; darkly stained

E.g. Barr bodies (inactive X) Calico Cats (shows that X inactivation israndom whether maternal or paternal)

C. Components:i. Isolated mitotic CH have (by mass): 20% DA, 70% Protein,


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ii. Concept of histone code = modification change inhistone structure/code change in chromatin structure activation/inactivation

Non-Histone Proteins (NHPs)remaining proteins; 50% = HPsa. Variable in number, amount, size, and charge (hard to study)b. Diverse functions = enzymatic, regulatory factors, structural, etc.

II. Structure of ChromatinUCLEOSOME = repeating units ofDA + Histones; primary, 1st order, basic building block, etc.

A. Experimental Evidence (Cornberg)i. Experiment = isolated nuclei orchromatin digested in varyingtimes/extents w/

ds-Dase (make double-strand cuts in DNA)ii. Observations (3 different typessize of DA,size of chromatin, appearance) =

Size of DNA (purified) usinggel electrophoresis after digestion forvarious time periods

Size of chromatin (DNA + its protein) usingsucrose gradientcentrifugation (smaller will stay up/at top)

Structure/appearance using electron microscope (EM)iii. Interpretations

Geometric series of sizes of DNA and of chromatin fragments,EM views,a. Sizes were 200, 400, etc. (fragment size linear relationship)

Fact that DNA in chromatin isOT totally digestible (plateaus at 50% w/DNA sizes of 146 bp), and

a. No matter how well digest, only is digested, rest is protected Remaining proteins are histone (H1) (in chromatin)

a. When remove H1 by subject to decrease in ionic strength, coreand linker DNA seen as separate elements

b. Shows ~beads strung togethersize of linking strand is 20 Observed dimensions, size of DNA, & equal mass Histones suggest = repeating unit has200 bp DA, equal mass of histones (200 bp), and hockey puck shape (11 nm

diameter X 5.7 nm high) = called UCLEOSOME (nuclear particle)

B. Model:i. core = 4 pairs of histones (H2a, H2b, H3, H4) w/ 146 bp DA (1 turns);

when linked to other nucleosomes and H1 present, have 2 turns (168 bp) of DNA If had been completely digested, 2 turns wouldnt be possible Core histones interacts thru theirtails =

a. Core organized into 4 hetero dimmers: 2 H2a-H2b & 2 H3-H4b. Dimerization done at C-terminal ends, which has -helices folded

into core of nucleosome (-terminalis long tail)ii. Spacer DA = bit variable, 40-60 bp, so repeat unit = 200 bp (linker DNA b/w

nucleosome generates these 200 bp)iii. X-ray crystallography studies showed thatDA is outside theprotein core

C. Higher Order Chromatin Structure?i. Still controversial, not yet settled, especially at higher levels

ii. Clear it involves more coilingand compactingto higher packaging ratiosiii. ext level = coiling of nucleosomes into regions that resemble cylinders

Stabilized byH1 histones Whether coiledzig-zagorsolenoidarrangement controversial (does so to

arrange thepacking)

All suggests = repeatingsubunit structure of

DA + Histones

DAHistone = non-covalent, ionicbond


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Diameter ~30 nm, so called 30 nm chromatin fibersD. Mitotic CH structure?

i. Even less agreement; involves coilingand organization (not random stuffing)ii. Reasonable hypothesis = protein scaffold (skeleton) organizes loops of 30 nm

coiled chromatin fibers materialiii. Experimental evidence?

Known =genes associatedw/ particular mitotic CH regions, so notrandom coiling(see this instaining patterns)

Experiment (Laemmli) = treat mitotic CH w/ high saltorpolyanion toremove all histones and most NHPs; compensate for charge of DNA sohistone proteins becomesolubilized

Observations =a. In light microscope:

i. Veryfuzzy expanded CH shapesii. Fuzzdestroyed by Dase, so DA = main structure

iii. Shape destroyed by Protease, but fuzz still present, soProtein = holds fuzz (DA) in shape; creates backbone

b. In electron microscope (EM):i. Huge 2 nm wide X 10-30 m long loops attached to

visiblefibrous scaffoldii. Use of enzymes showed:

1. Loops =DA (very skinny; 30 nm filament)2. Scaffold =Protein (organizes 30 nm filament; b/c

not extractable, so stay there and make backbone)c. SDS protein gels (PAGE):

i. Showed less than 10 HPs in the protein scaffold (only 2common ones, one of them = toposiomerase II)

ii. Toposiomerase IIunwinds DA; regulates of coilingIII. Role of Chromosome Structure in Transcriptional Activity

A. When chromatinfully compacted(heterochromatin), no or very little transcriptioni. Constitutive (always on) = as around centromeres, telomeres w/ few or no

genes, and repeated sequences Highly repeated, never unwinds, so inactive and always hetero

ii. Facultative = regulated, restricted to specific regions/times Extreme caserandom inactivation of an X CH in females (Barr Body)

So, forfull activation/transcription, need to uncompact or unwindcondensed chromatin and nucleosomes

B. Mechanisms:i. Histone Code = modification of histone-terminal tails which stick out from

nucleosome core (reversible & diff. specific enzymes used for different histones) Types:

a. Methylation (Histone + DA)of specific lysine, arginine A.A.b. Acetylation (Histone)oflysinec. Phosphorylation (Histone)ofserine

Specific enzymes involved: can be specific for particularhistones andA.As (e.g., histone methyltransferase for lysine9 in Histone H3)

Reversible: but w/ different enzymes (e.g., histone acetyltransferase(HAT) and histone deacetylase (HDAC))

DA coiled w/histones

(nucleosomes) 30

nm chromatin fibersloops protein

scaffold organizesloops make up parts

ofmitotic CH

50% of DA never

gets digested; why?

= b/c even the 50%that is digested willbe lot of coiling;also, theH1 willcome offif digest

completely

CompactDA =Inactive &

Open it= Active

hetero = highly methylated,

eu = not [HISTOE

ACETYLATIO= oppositeeffectthan methylation;

prevents CH fibers fromfoldinginto compact structures

eu; access of regions ofDNA to interacting proteins transcription; added to lysine

byHATs]


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Results of these modifications:a. Charge of histone tails changes and so tightness of histone-DNA

binding changedb. Create binding sites for other proteins/complexes that affect

chromatin structurec. Can be eitheractivation orrepression depending on type and

location of modification (depending on histone and its location)i. Inhibitorymethylation of H3lys9 recruits a protein

(HP1) important in forming/maintaining heterochromatinw/ other proteins

ii. Activationacetylation; HATs act as coactivators fortranscription inducing less compaction, more open andaccessible DNA for transcription factors

ii. Specific small non-coding RAs can target specific DA sequences and affectchromatin (new observations and so less well understood)

iii. Chromatin Remodeling Complexes (CRCs) = remodelingof histones Composed of variousspecific proteins (e.g. SWI, SNF or actin family) Use energy from ATPto alter chromatin/nucleosome structure to openspecific DNA sequences for binding by transcription factors

iv. DA methylation (on cytosine) = often a tag forrepression Specific enzymes (DNA methyltransferases) onspecific Cs onDA

a. Oftensymmetrically on both strands in CpG dinucleotide (non-random abundance)

b. Newly synthesized strand will have methylation atsame place Usually associated w/ recruitment of other proteins that induce/maintain

repression (keeps entire CH repressed) Maintained thru cell division, since after DNA replication hemi-

methylated converted to fully symmetrical methylation

(Note: not every organism uses this methylation mechanism; not inyeast,nematodes = they do acetylation and methylation)

IV. Eukaryotic Chromosome ReplicationA. Similar, yet a bit different fromprokaryotes (eukaryotic DNA not circular)

i. Semi-conservativeii. Similar types of enzymes (5 3 polymerases (replicases, repair, etc.), primase,

helicases, ligases, etc)iii. Discontinuous synthesis on lagging strand(Okazaki fragments ---- butsmallerin

eukaryotes)iv. Bidirectional replication w/ forks

B. Evidence (Hubermann and Riggs)i. Experiment (Part #1) =pulse-labeltissue culture cells 20 min w/H3-thymidine

(tagged DNA made during one short labeling period) Isolate DA andspread overmicroscope slide Autoradiography w/ film (exposed by isotope)

a. Allows one to see resolution of DAb. B/c cant see isotope until its exposed on film = autoradiography

ii. Observations = ( )( ) = 15-30 m tracks Spaces b/w show multiple replicons On the same piece of DNA, >1 replication forks going on

Acetylating TATA

Box Replication

Active CHhaveacetylated histones

Used tissue culturethatgrow rapidlyand label that waseasy to see under

microscope

Properties of chromatin

that depend on

modification: (1) degree ofcompaction (whether region

is hetero or eu & (2)likelihood that gene/cluster

will be transcribed

hetero = highly

methylated, eu = not

[HISTOEMETHYLATIO= makes histone capable ofbinding w/ high affinity to

protein w/ particular domain higher formation of

interconnected nucleosomes

higher order ofincreasedcompaction = hetero]


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iii. Interpretations = rate is to 1 m/min (2000 bp/min), which isslowerthan inprokaryotes such as E. coli (50,000 bp/min)

Probably b/c have to replicate chromatin, and not just naked DAiv. Experiment (Part #2) =pulse-label30 min, but then chase for 45 min w/

excess unlabelled thymidine After 20 min, allowed growth of DA for 45 min If unidirectional gradient, also in one direction; if bidirectional gradient,

goes in opposite directionv. Observations =gradients of silver grains going in both directions & see forks

DNA made during chase periodis decreasing in radioactivity b/cunlabelled thymidine is replacinglabeled thymidine

vi. Interpretations = Replication is bidirectionaland uses forks like prokaryotes Eukaryotes have many replicons along same DNA, compared to

prokaryotes which have one Each replicon has an origin in middle and terminators at each end Size of eukaryotic replicons (measures from origin to origin) averages

from 7-30 m (sosmallerthan E. colis 1360 m circle)C. Significance/Importance for Many Small Replicons for Eukaryotes

i. Cells can replicate all of their DA in ashort time (i.e. huge eukaryotic DNAneeds >1 replicon sinceslow)

ii. Why so many replicons?b/c ifone per CH, would take lot of time so manyparts need to be replicated together

iii. Are all replicons working simultaneously?O b/c that would be very fast(allwould be done in


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Cant finish replicatinglittle gap left when RNA primer removed fromlagging strand, since no free 3 OH group for DNA pol to use to begin

So, ends would havesingle-strand overhanging ends and not have doublestranded blunt ends and would getshorterw/ each replication

iii. Solution to this problem:

Prokaryoteshave circular DNA for genome/CH, so no free ends Eukaryoteshavespecial DA at telomeres (ends) and special

telomerase enzyme to replicate this DNAa. Structure of telomere DA = highly repetitive units of 5-6 ntse.g., 5 (TTGGG)n 3 in tetrahymena & 5 (TTAGGG)n 3 in humans

(repeated many times as buffer toprotectthe gene of importance)b. Overhanging 3 end, tucked in looptake ends that is

overhangand put in loop to protect the telomere endc. 100s-1000s of copies in each telomere

Telomeraseunusual enzyme composed ofprotein + RAa. RA = servers as template (primer) and telomerase enzyme

(protein) copies this RNA sequence into DNA

b. Protein = serves as enzyme to do replication, polymerization, andforming covalent bonds

c. Thus, it has reverse transcriptase activity analogous to RNAretroviruses like HIV, but contains its own template

Mechanismrepetitive inchworm synthesis of these 5-6 nucleotidesingle-strand DA units to overhanging 3 endof CH DNA, which thengets replicated by usual system

a. Goes overstrand and extends only the particular strand itsworking on

b. What control how many copies to add not fully understoodc. Other proteins clearly importantsince in cells w/ telomerase, size

approximately constant Amountof telomerase is important

a. Recent work suggests that in aging cells, the number ofcopies/size of telomeric DNA goes down b/c level of telomerasedecreases/gone in somatic cells after differentiation

b. Idea = eventually the important CH regions getshortenedandthen genetic defects/problems (Note: notin gamete cells)

(One reason for aging?)c. Transformed cancerous cells have detectable levels of

telomerase (compared to normal cells)d. So, one important part of why cancer cells have abnormal growth

controlmay involve this telomere replication activity in addition

to other mutated genes (e.g. oncogenes like RAS)e. So, trade off/balance b/wgrowing oldandprobability of

getting cancer? So, only telomerase DNA uses reverse transcriptase

a. Cells go thru crisis get transformedand become immortalcancerous (telomerase activity is increasing)

b. Cancer is hetergenousresponse = no longer respond to bodysignals to stop dividing

5 end of new

strand is gettingshorterwhile 3

end of old strand isgetting overhung

Telomere DNA highlyconserved; fixed in all

organisms (e.g., TTAGGG= same in all vertebrates,

not just humans)

Telomerase =RAdependent,DA

polymeraseTemplate is within

enzyme

Overhangonly on 3strand b/c thats where

the special enzymeworksadds on 3 when

not in loop

In normal cells, rapidlydividing cells have a lot of

telomerase activity; however,cells in brain, liver, etc. havealmost nonexistent telomeraseactivity (analysis of this: oldercell = telomerase activity =

telomeres get shorter)

RA pol can dow/o 3 OHbut not

DA pol

Chromosomes and Replication

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