L16-17: Dna Topology I-Ii

Front 1

Dna length

Back 1

2 meters (6 feet)

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nucleus

Back 2

few micrometers

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Topology

Back 3

spatial properties that are preserved under continuous deformations of objects

Front 4

DNA is packaged into

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chromosomes

Front 5

DNA must be highly

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compacted: compaction ratio about 10,000

Front 6

Supercoiling

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coiling of a coil

Front 7

Distortions caused by

Back 7

torsional stress

Front 8

DNA supercoiling is result of

Back 8

topological property of DNA

Front 9

Topological property only applied if

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both strands remain covalently intact & tension is stored: closed-circular DNA is good example

Front 10

If a break occurs in any strand

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torsional strains & supercoils are removed

Front 11

Supercoiling in eukaryotic chromosomes

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Chromosomal DNA cannot freely rotate along its length & are organized into loops whose ends are restrained very much like a circular DNA mlc

Front 12

Euk chromosomes are not closed DNA circles

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but have topologically restrained regions

Front 13

Deviation from duplex B

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form induces supercoiling

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Duplex B form DNA

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1 turn every 10.5bp

Front 15

DNA supercoiling tends to form when

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2 strands of circular DNA duplex are wound around each other more or less than once every 10.5 bp

Front 16

Cellular DNA is

Back 16

underwound & negatively supercoiled

Front 17

Linking number

Back 17

topological property useful to measure degree of supercoiling

Front 18

LK = number of times

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one strand of DNA winds around the other

Front 19

LK naut

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relaxed state in B form DNA = number bp divided by 10.5 bp

Front 20

LK only applied if both strands remain covalently intact

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closed-circular DNA: if break occurs in any strand, there is no LK

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Twist & writhe must equal

Back 21

linking number

Front 22

TW

Back 22

twist number (helical turns)

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Wr

Back 23

writhing number: large distortions of long axis of double helix

Front 24

Superhelical density

Back 24

# turns removed or added relative to # present in relaxed state

Front 25

Superhelical density equation

Back 25

change LK/ LK naut

Front 26

In cells, superhelical density is

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5-7%, that is -.05 to -.07 (negative means underwinding of DNA)

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Cells maintain DNA in underwound state to facilitate

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compaction & strand separation

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plectonemic

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40% of length -> not enough compaction

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spiral

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much more compaction, found in Euk cells

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strand separation

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allowing access to information (replication, transcription)

Front 31

Different segments of genome are

Back 31

replicated or transcribed @ different times

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Topological state needs to be

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dynamic & highly regulated

Front 33

Topoisomerases

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enzymes that can regulate topological state by changing linking number

Front 34

Circular DNAs w same # base pairs

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but differ in linking number can be separated by gel electrophoresis

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Topoisomerases can relax

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supercoiled DNA

Front 36

Topoisomerase 1

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increases LK by steps of 1

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Topoisomerases catalyze a 3-step process

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1. Cleavage of DNA strand 2. Passage of segment of DNA through break 3. Resealing of DNA break

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Topoisomerases are ___ enzymes

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nicking-closing

Front 39

Type I topoisomerases cleave

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one strand & do not require ATP

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Type II topoisomerases

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change LK in steps of 2, cleave both strands, require ATP

Front 41

Topoisomerases form a covalent

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protein-DNA link

Front 42

Tyrosine residue in active site

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of topoisomerase attacks phosphodiester bond in backbone of target DNA, resulting in covalent 5' -phospho-tyrosyl DNA protein linkage

Front 43

NO ATP needed

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for this rxn (in case of topoisomerase II, ATP hydrolysis used for conf changes)

Front 44

Mechanism of bacterial T1 topoisomerase

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1st conf change (closed-to-open) promotes passage of strand

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Second conformation change

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open-to-closed brings cleaved ends back together

Front 46

Model for type 2 topoisomerases is similar

Back 46

but involves dimers (2 reactive tyrosines)

Front 47

DNA replication generates major

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topological problem

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DNA replication requires

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separation of strands

Front 49

Further continuous strand separation requires

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rotation of rest of DNA double helix

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Topoisomerases travel in front of areas of strand separation

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allowing rotation of only a short length of helix, relieving tension

Front 51

Conclusion: topoisomerases are

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ESSENTIAL for DNA replication

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Topo I & II from Euk

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remove both positive & negative supercoils

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Topo I from bacteria

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removes negative but not positive supercoils

Front 54

Bacteria have TII topo that

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relax positive supercoils & introduce negative supercoils

Front 55

Prokaryotes have special topoisomerase

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that introduces supercoils: DNA GYRASE

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DNA gyrase

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keeps bacterial genome in negative supercoiled state

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DNA gyrase introduces

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negative supercoils & relax positive supercoils

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Topoisomerases are

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drug targets

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Without topoisomerases

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cells cannot replicate DNA or transcribe genes

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Antiobiotics - rationale

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some bacterial & fungal topoisomerases are significantly different from mammalian topoisomerases

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Example: Quinolones (cirpofloxacin)

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target DNA gyrase & topoisomerase IV

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Ciprofloxacin used to treat

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anthrax & some urinary infections

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Cancer therapy: rationate

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highly proliferating cancer cells are more sensitive to topo inhibitors

Front 64

Campothecin analogs (irinotecan & topotecan)

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inhibit eukaryotic type I topoisomerase

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Irinotecan

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used to treat colorectal cancer & ovarian cancer