Lecture 17 • Enzymes that break and ligate DNA & RNA backbones; DNA topology

Key learning goals:• Understand pyrophosphatase as a model for

phosphoryltransfer reactions• Understand the classes of nucleases: exonucleases,

endonucleases, restriction enzymes.• Understand ligase enzymes.• Understand the problems in packaging DNA into a

bacterium, nucleus, or virion.• Topology: understand linking number, twist, and

writhe.• Understand topoisomerase enzymes (type Ia, Ib, II),

and how they di!er from helicases.• Understand why gyrase is a good antibiotic target.• Understand why human Topo II’s are chemo targets.

dsDNA ssDNA nucleotides

dA

dC

dG dU

Optical properties of nt’s, ssDNA, dsDNAA260 nm

The conjugated !-electron systems of the purine & pyrimidine bases absorb strongly in the UV band.

The excited states of two interacting molecules can be described as linear combinations of the excited states of each.

In certain geometries, some of the absorption strength in the near-UV moves to bands at higher energies.

This is the hyperchromic e!ect.

100

80

60

40

20

0

% D

enat

ured

110100908070Temperature / oC

40 50 70% GC60

The ratio of A:T to G:C base pairs influences Tm

?

1. As DNA melts, what happens to the UV absorbance?

2. As the fraction of A:T base pairs rises, what happens to Tm ?

3. What might we expect the base compostion of an arctic fish to look like vs. a tropical fish?

4. How can we best explain the e!ects of base compostion on Tm ?

A260 nm

?

?

DNA and RNA hybridization — base stacking energies depend on the sequence!

h"p://sandwalk.blogspot.com/2007/12/dna-denaturation-and-renaturation-and.html

Helicases are motors that hydrolyze ATP to melt DNA and RNAHelicases hydrolyze the

terminal (γ) phosphate on ATP:

ATP —> ADP + Pi

Helicases do not break the backbone!!

E. coli alone has twelve DNA helicases and more RNA helicases; DnaB unwinds DNA for replication.

DNA helicase

Helicases are motors that hydrolyze ATP to melt DNA and RNAHelicases hydrolyze the

terminal (γ) phosphate on ATP:

ATP —> ADP + Pi

Helicases do not break the backbone!!

E. coli alone has twelve DNA helicases and more RNA helicases; DnaB unwinds DNA for replication.

An RNA helicase would “unzip” (= denature or melt) this structure

single strands

bulge

internal loop

hairpin

A-form double helix

A

A C A

G A

CGG GCC

UCCU

AGGA CGU

GCA

A A U

A A

G

GAUGG

CUACC

GGAAC

AUGC U

AGCA CCUUG A

G G C

A T

A

A

γ β α

anhydridebonds

phosphoesterbond

5´

3´

in DNA and RNA,adjacent nucleotides

are linked thoughphosphodiesters

C

G

T

A

HO-CH2 O

H2N-C

C

C

HN

N

N

CH

C

O

N

O

O

O P O CH2 O

O

C

N

N

CH C

CH

NH2

NH2

C

C N

N

N

CH

C

N HC

O

O

O P O CH2 O

O-PO32

O

O

O P O CH2 O

N

C C O

HN

CH C O

CH3

5´

3´

5´

3´

DNA and RNA backbones are synthesized and broken through phosphoryltransfer reactions

A note on ATP equivalents in metabolic accounting

γ β α

5´

3´

1 2 3

position where initial hydrolysis occurs:

# of ATP equivalents:

This could be A, G, C, etc.

Count the # of phosporylations (by Nu kinases, F1F0 ATPase, etc.)

required to get back to the NTP state.

Inorganic pyrophosphatase catalyzes irreversible PPi hydrolysis

PPi + H20 2Pi + heat ("G° = –19 kJ)

The simplest phosphoryltransfer reaction (transfer to water)

PPi’ase accelerates reaction # 1010

Requires 3 or 4 divalent M2+ ions (usually Mg2+ but other metals can substitute)

E. coli PPi’ase is this exceptionally pre$y homohexamer

Eukaryotic PPi’ases are homodimers

Active sites are very similar; typically, 13 of the 17 active site residues are conserved

Pyrophosphatase catalytic site (yeast)

Acid/base catalysis; no covalent enzyme-bound intermediate

Acidic residues and H2O molecules coordinate M2+

P2 is electrophile

SN2-like a$ack on P2(interchange mechanism)by base [hydroxide]coordinated by M2+ ions

Transition state: penta-coordinate center on P2.

acid [water]

Modified from Heikinheimo & al. 1996, Structure 4:1491

Nucleases — phosphoesterases that cleave DNA and RNA

Substrates: DNAses / RNAses

General reaction: Phosphoester + H2O acid (R-Pi) + alcohol (R-OH)

• M2+ cofactor required (usually Mg2+)

• Some nucleases leave 3´-Pi while others leave 5´-Pi

P

C

5´P

T

5´P

G

5´

3´ 3´ 3´

P

C

5´P

T

5´P

G

5´

3´ 3´ 3´

+

P

C

5´P

T

5´P

G

5´

3´ 3´ 3´

+

or

Nucleases — phosphoesterases that cleave DNA and RNA

Exonucleases chew away on free endsTwo main types, chew in opposite directions

• 5´–3´ enzymes require free 5´end on substrate• 3´–5´ enzymes require free 3´end.

Endonucleases cut in the middle of a strand or duplex

• Some are relatively non-specific (will cleave any sequence)• Site-specific nucleases cleave only at specific sequences• Some cut a single strand; others cut both strands.

Clint Eastwood cuts it like an endonuclease in The Eiger Sanction

Restriction enzymes: site-specific dsDNA endonucleases

Bacteria use these enzymes to defend against viruses and other sources of foreign DNA

The restriction enzyme wraps around the DNA and scans for specific recognition sequences.

At these “restriction sites” the enzyme cleaves both backbones.

! CH3

CH3

x

!

!Restriction site:

often a 4, 6, or 8 bp inverted repeat

In nature, restriction enzymes are found in matched pairs with DNA methyltransferases (DMT).The DMTs methylate DNA at identical recognition sequences. This protects the bacterium’s own DNA from cleavage by the nuclease.Thousands of di!erent restriction-methylation systems have been identified. Hundreds of di!erent restriction enzymes are used in biotech.

Example: EcoR1, a type II restriction endonuclease

5’…GAATTC…3’3’…CTTAAG…5’

5’…G3’…CTTAAp

pAATTC…3’ G…5’+EcoR1, Mg2+

2H2O+

EcoR1 homodimer and substrate.Mg2+ ions not present in crystal.

PDB 1RVA

EcoR1 and product.For clarity, only one of the two

EcoR1 subunits is shown. PDB 1RVC

DNA Ligase seals ss nicks

1. Ligase active site Lys residue forms covalent activated intermediate with NAD or AMP

3. Nucleophilic a"ackby 3´-OH seals nick, regenerates enzyme + AMP

2. Adenosine-5´-diphosphate a"ached to 5´ end of nick. Note 5´-5´-diphosphotriester!*

* A couple of lectures from now you’ll see a similar, unusual 5´-5´ linkage @ the 5´end of eukaryotic mRNA molecules!

DNA is packaged into small volumes

Volume of mammalian nucleus*: 0.1 pL = 10–16L

Human genome: 6 x 109 bases

What is the net charge of the DNA in a human nucleus, in units of moles of charge per L (M)?

6 x 109 Pi 10–16L

x 1 mol6 x 1023 Pi

= 10 mol Pi L

negative charge: ~ 10 M…DNA backbone ~10 N phosphoric acid!…requires ~10 N positive counterions

* J Biol Chem 2006, 281:8917-8926

DNA topology: loops of DNA are anchored to chromsomal sca!olds.

DNA topology: supercoiled loops of DNA are anchored to chromsomal sca!olds.

supercoiled DNA supercoiled protein

Bacterial chromosomes and plasmids are closed, circular, double-stranded DNA — and generally supercoiled.

DNA is torsionally sti!.It resists being untwisted or over-twisted. DNA supercoiling is a manifestation of torsional strain.

increasing supercoilingrelaxed

DNA topology: writhe (W) and twist (T)

DNA topology: linking number (L)

L = T + W (absolute)!L = L – L0 (relative to relaxed B-DNA)

L0 !L = -3

!T = !W =

00

–30

–2–1

–1–2

0–3

!L = (!T + !W)

DNA topology: spooling of DNA onto histones removes negative twist, increases writhe

DNA topology: removal of histones histones makes twist more negative, facilitating local melting

DNA intercalating agents decrease twist

L = T + W (absolute)!L = L – L0 (relative to relaxed B-DNA)

Topoisomerases: enzymes that modify linking number (L)

Must cleave one or both backbones

Must not allow cleaved intermediates to di!use away

Must re-ligate broken backbones

•Relaxes negatively supercoiled DNA. •Can interlink (catenate) circles of ssDNA. •Covalent intermediate: Tyr on Topo I transiently linked to the DNA through phosphodiester bond. • Does not need ATP.

Topoisomerase IA

•Relaxes negatively supercoiled DNA. •Can interlink (catenate) circles of ssDNA. •Covalent intermediate: Tyr on Topo I transiently linked to the DNA through phosphodiester bond. • Does not need ATP.

Topoisomerase IA

Professor Wim Hol, UW Biochemistry

Relaxes highly supercoiled DNARelaxes negative OR positive supercoilsDoes not interlink ssDNA circlesControlled rotation mechanismCovalent intermediate, as in Topo IADoes not need ATP

Topoisomerase IB

Relaxes highly supercoiled DNARelaxes negative OR positive supercoilsDoes not interlink ssDNA circlesControlled rotation mechanismCovalent intermediate, as in Topo IADoes not need ATP

Professor Wim Hol, UW Biochemistry

Topoisomerase IB

Passes one strand of dsDNA through another.• Breaks and re-ligates both strands of one duplex• Symmetric dimer• Consumes ATP, releases ADP and Pi• Can interlink (catenate) two dsDNA circles

Berger Lab, UC Berkeley

Topoisomerase II

A specialized type II enzyme that seems to be present only in bacteria.

It is the only enzyme known that can introduce negative supercoils.

This means that the enzyme does mechanical work!

(what’s the d! of mechanical work?)

Gyrase – a bacterial Topo II

That’s 100 turns of the helix per second— DNA unwinds at almost 10,000 r.p.m. !!!

Topoisomerases are essential for DNA replication

DNA helicaseDNA topoisomerase II(in bacteria: gyrase)

103 bp/s

These important antibiotics rather selectively inhibit gyrase, so they kill bacteria but not eukaryotic cells

These Topo II inhibitors are important anti-cancer drugs.

Topo II enzymes: antibiotic and chemotherapy targets