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 differ from helicases. • Understand why gyrase is a good antibiotic target. • Understand why human Topo II’s are chemo targets.
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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
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
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
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