Lehninger PPT Ch24

Download as ppt, pdf, or txt
Download as ppt, pdf, or txt
You are on page 1of 61

24| Genes and Chromosomes

© 2013 W. H. Freeman and Company


DNA Supercoiling

• DNA in the cell must be organized to allow:


– Packing of large DNA molecules within the cells
– Access of proteins to read the information in DNA
sequence
• There are several levels of organization, one of
which is the supercoiling of the double-stranded
DNA helix
Helical Supercoils
Supercoiling is the coiling of a coil

• Non-supercoiled DNA is called relaxed


• Many circular DNAs are supercoiled
• Supercoiling has great influence on transcription and replication of DNA
• Supercoiling can be highly regulated
Closing DNA in a loop introduces
supercoiling
The Effects of Replication and
Transcription on DNA Supercoiling
Most cellular DNA is underwound

• Normal B-form, relaxed DNA: 10.5 bp/turn


• Closed circular DNA is rarely relaxed
– Strain induces supercoiling
– Strain is due to fewer helical turns (underwinding)
– Underwinding makes later separation of the strands
easier
• Linear DNA is underwound with the help of
proteins to prevent strands from rotating
Relaxed and Increasingly
Supercoiled Plasmid DNA
Effects of DNA Underwinding
Linking number (Lk) describes supercoiling

• In circular DNA, changing the helical turns requires breaking a strand transiently
• Linking number in relaxed DNA:
Lk = #bp  #bp/turn

Example: relaxed circular dsDNA of 2100 bp in the B form (10.5 bp/turn) has
Lk = 2100 bp  10.5/turn = 200

Lk is an integer for closed-circular DNA and is (+), reflecting a right-handed


helix.
Lk is the number of times a strand
penetrates a surface
Lk Applied to Closed-Circular DNA
Changes in Lk are useful for
describing supercoiling
• Consider a relaxed DNA of 2100 bp:
Lk0 = 200

• Underwind by removing two helical turns:


Lk = 198
Lk = 198 – 200 = –2
Superhelical Density, 
Negative and Positive Supercoils
Linking number can be broken down
into Twist (Tw) and Write (Wr)

Lk = Tw + Wr
•Twist (Tw) is the # twists or turns of the
helix
•Writhe (Wr) is the # coils
– Typically a negative value
Twist and Writhe
Underwinding Facilitates Additional
DNA Structural Changes

• Helps to maintain structure of cruciforms at


palindromes (next slide)

• Cruciforms rarely occur in relaxed DNA

• Facilitates formation of stretches of left-


handed Z-form
Cruciforms
Topoisomers are DNAs that differ
only in linking number
• Same # bp, same sequence but different degree of supercoiling

• Conversion between topoisomers requires a DNA strand break

• Note that negatively supercoiled DNA (more compact) travels faster in


agarose gel electrophoresis experiment than relaxed or nicked DNA
Topoisomers in Electrophoresis
Topoisomerases are enzymes that
change Lk
• These enzymes are required for DNA unwinding and
rewinding during transcription and replication
• Two major types:
– Type I – make a transient cut in one DNA strand
– Type II – make a transient cut in both DNA
strands, change Lk in steps of 2.
The Topoisomerases I–IV of
E. coli
• Topo I and III are Type I
– removes negative supercoils to relax DNA
– Use single-strand breaks
• Topo II is called DNA gyrase
– Introduces supercoils
– Uses ATP and double-strand breaks
Type 1 Topoisomerase
Eukaryotic topoisomerases include
Topo I, II, II, IV
• Topo I and III are Type I (as in E. coli)
• Type II topoisomerases include two
subfamilies—Type IIA and Type IIB
– Can relax both positive and negative supercoils
Mechanism of a Eukaryotic
Type IIA Topoisomerase
Topoisomerases are targets for
antibiotics
• Coumarins (novobiocin, coumermycin A1)
– inhibit bacterial type II topoisomerases from binding
ATP

• Quinolones (nalidixic acid; ciproflaoxadin, Cipro)


– Inhibit the last step, which is resealing the DNA strand
breaks
– Wide-spectrum and mostly selective for bacterial
enzymes
Topoisomerase Inhibitors Used as
Antibiotics
Topoisomerase Inhibitors Used as
Chemotherapy Agents

• Targets cancer because most rapidly growing cells


(tumors, others) express topoisomerases
• Eukaryotic Type I topoisomerase inhibitors
– Captothecin, irinotecan (Campto), topotecan
(Hycamtin)
– Trap the enzyme-DNA complex in its cleaved state
• Eukaryotic Type II topoisomerase inhibitors
– Doxorubicin (Adriamycin), etoposide (Etopophos),
ellepticine
Topoisomerase Inhibitors Used as
Chemotherapy Drugs
Supercoiled DNA forms Plectonomic or
Toroid/Solenoid structures
(or a combination)
• Plectonomic
– Seen in plasmids
– Involves a right-handed superhelix with terminal loops
• Toroid/Solenoid
– Used in chromatin
– Involves tight left-handed turns
– Can resemble a garden hose on a reel
– Provides more compaction
Plectonemic Supercoiling
Plectonomic and Solenoidal
Supercoiling
Eukaryotic chromosome structure
changes over the course of a cell cycle

• Nondividing state (G0) and Interphase


(Gap1, G1; Synthesis, S; and Gap2, G2):
– Chromatin is amorphous, randomly dispersed
• Prophase of mitosis
– Chromosomes become condensed, pairs of
sister chromatids form
DNA Packing into Chromatin
• Chromatin consists of fibers of protein and DNA and small amount of RNA
• DNA associates tightly with proteins called histones
– Small proteins with lots of basic (Lys, Arg) residues
– Are modified by methylation, acetylation, ADP-ribosylation, phosphorylation, glycosylation, sumoylation, ubiquination
• DNA and protein are packed into discrete units called nucleosomes
Changes in Chromosome Structure
During the Cell Cycle
Nucleosomes consist of DNA wrapped
around histones
• DNA of length 105 m  fits into nucleus of
diameter 5–10 m
• Partial unfolding reveals “beads on a string”
– Beads are ~146 bp of DNA wrapped around 8 histones
(the “core”)
– Forms left-handed solenoid
– String is “linker” DNA of ~54 bp bound to histone H1
– Amino-terminal tails of histones stick out, form sites for
covalent modification, and form important contacts
between nucleosomes
Nucleosomes
Nucleosomal DNA is underwound

• Wrapping DNA around the histone core


requires removal of one helical turn
– The underwinding occurs without a strand
break, so a compensatory (+) supercoil forms
– This (+) supercoil is relaxed by a
topoisomerase, leaves DNA with net negative
supercoil
DNA Wrapped Around a Histone
Core
Front and Side Views
of Histone Amino-
Terminal Tails
How Amino-Terminal Tails Interact
between Nucleosomes
Histone binding depends on DNA
sequence
• Histone binding is not random
• Occurs more often at A-T–rich regions
• Staggering AA, AT or TT at 10 bp intervals (phased with pitch of helix) narrows the minor groove, bends the DNA
 facilitates its binding around the histone core
Effect of DNA Sequence on
Nucleosome Binding
Nucleosomes then assemble into
higher-order structures
• Nucleosome formation compacts DNA seven-
fold…but overall compaction is >10K-fold!
• Next level of structure: 30 nm fiber
• Occurs in regions where sequence-specific,
non-histone proteins bind
• Two models in Figure 24-29: solenoid model
and zigzap model
The 30 nm Fiber
Higher-level structures depend on
chromosomal scaffold
• Higher-order structures are not well understood
• May vary between chromosomes, regions, even
moment of time
• Appears to involve a loop of DNA (maybe with
related genes) associating with a scaffold of
proteins
– Includes Topo II and SMC proteins
• SMC = Structural Maintenance of Chromosomes
Loops of DNA Attached to a
Chromosomal Scaffold
Compaction of DNA in a Eukaryotic
Chromosome
Condensed chromosome structure is
maintained by SMC proteins

• SMC proteins have five domains


• N and C domains form ATP-binding site
• Two major types
– Cohesins – help link sister chromatids
– Condensins – help chromosomes to condense;
create positive supercoils
• See Figure 24-32
Structure of SMC Proteins
Bacterial DNA is organized into
nucleoids
• Can occupy much of cell volume
• DNA attaches to plasma membrane
• Scaffold-like structure organizes the circular DNA
into ~500 looped domains
• DNA binds to proteins transiently
– Example: Protein HU
Possible Role of Condensins
Roles of Cohesins and Condensins
in the Cell Cycle
E. coli Nucleoids
Looped Domains of the E. coli
Chromosome
Chapter 24: Summary
In this chapter, we learned:
• Genetic information of the cell is encoded in the nucleotide
sequence of one or several DNA molecules
• The protein-coding regions represent only a small fraction of the
total DNA
• Telomeres and centromeres regulate cell division
• Bacterial DNA is usually supercoiled for efficient packing
• Eukaryotic DNA is wound around positively charged histones
• Higher-order organization of chromatin likely involves coils upon
coils upon coils

You might also like