genome organization in prokaryotes, mechanism of supercoiling, Role of topoisomerases,

1. Prokaryotic Genome Organization,
DNA Supercoiling And Role Of
Topoisomerases
Presentedby,
MANOJ BS
Ph.D.Scholar
J-17-D-23-BS

2. Contents:
ProkaryoticGenomeOrganization
DNASupercoiling
 Definition.
 Mechanism.
 Types of supercoiling(Positive and Negative).
 Linking number.
 Functions of supercoiling.
 Methods of measuring supercoiling.
 Supercoiling in Prokaryotes and Eukaryotes.
Topoisomerases
 Role of topoisomerases in supercoiling.
 Different types of topoisomerase.
 Functions of topoisomerase.

3. INTRODUCTION
• The term “prokaryote” means “primitive nucleus”. Cell in prokaryotes have no nucleus. The
prokaryotic chromosome is dispersed within the cell and is not enclosed by a separate membrane.
• Much of the information about the structure of DNA has come from studies of prokaryotes,
because they are less complex (genetically and biochemically) than eukaryotes.
• Prokaryotes are monoploid = they have only one set of genes (one copy of the genome).
• In most viruses and prokaryotes, the single set of genes is stored in a single chromosome (single
molecule either RNA or DNA).
• Prokaryotic genomes are exemplified by the E. coli chromosome.
• The bulk of the DNA in E. coli cells consists of a single closed-circular DNA molecule of
length 4.6 million base pairs.
• The DNA is packaged into a region of the cell known as the nucleoid.

7. E.g: E. Coli
• 89% coding
• 4285 genes
• 122 structural RNA genes
• Operon: Polycistronic transcriptom units
• Haploid circular genome
• Usually asexual reproduction
• Transcription and translation take place in
the same compartment.

8. DNA SUPERCOILING
DNA supercoiling refers to the over- or under-
winding of a DNA strand, and is an expression of
the strain on that strand.
Asuper coiled form of DNA is the one in which
the double helix is further twisted about itself,
forming a tightly coiled structure.

9. Mechanism
A double helix of DNA undergoes
additional twisting in the same direction as or in
the opposite direction from the turns in the
original helix. Supercoiling results when DNA is
subject to some form of structural strain. A
strain is introduced in the DNA to induce
supercoiling.

10. Original DNAform
The most common double helical structure found in
nature is B-DNA in which the double helix is right-handed
with about 10–10.5 base pairs per turn
Underwound DNA form
Underwound state occurs when the DNA has
fewer helical turns than the normal B-form.

11. Cells maintain DNAin an underwound state
to;
1)Facilitate its compaction by coiling.
2)Enable the enzymes responsible for DNA
metabolism to separate DNA strands.
This state however causes the molecule to be
thermodynamically strained. The strain is accommodated
by coiling of the axis of DNA on itself to form supercoil.

12. COILING SUPERCOIL

13. NEGATIVE SUPERCOIL (LEFT HANDED)
Negative supercoiling involves twisting
against the helical conformation (twisting in a left-handed
fashion), which preferentially underwinds and "straightens"
the helix at low twisting stress, and knots the DNA into
negative supercoils at high twisting stress
POSITIVE SUPERCOIL (RIGHTHANDED)
Positive supercoiling of DNA occurs when the
right-handed, double-helical conformation of DNA is
twisted even tighter (twisted in a right-handed
fashion) until the helix begins to distort and "knot."

15. Linking number
Numerical expression for degree of supercoiling
A. Equation Lk=Tw+Wr
B. L:linking number, # of times that one DNAstrand windsabout
the others strands, is always an integer
C. T:twist,# of revolutions about the duplex helix
D. W:writhe, # of turns of the duplex axis about the
superhelicalaxis by definition the measure of the degree of
supercoiling
E. Specific linking difference or superhelical density=∆Lk/Lk0

23. Functions of supercoiling
 Genome packaging
 Gene Expression
 DNA/ RNASynthesis

24. Methods for measuring supercoiling
Based on how compact the DNAis
A. Gel electrophoresis
i)1 dimensional
ii)2 dimensional
B. Density sedimentation

26. Supercoiling in prokaryotes
In prokaryotes, plectonemic supercoils are predominant, because
of the circular chromosome and relatively small amount of genetic
material.
This structure is called the FOLDED GENOME.
Within the folded genome, the chromosome is organized into
domains or loops, each of which is independently negatively
supercoiled.
A dimeric protein – HU condenses DNA and wrap it in bead like
structure.

28. Supercoiling in Eukaryotes
 In eukaryotes, DNA supercoiling exists on many levels of both
plectonemic and solenoidal supercoils.
 The solenoidal supercoiling proving most effective in compacting the
DNA.
 Solenoidal supercoiling is achieved with histones to form a 10 nm
fiber.
 This fiber is further coiled into a 30 nm fiber, and further coiled upon
itself numerous times more to form chromatin and eventually
chromosome.

29. TOPOISOMERASE
Topoisomerases are enzymes that regulate the
overwinding or underwinding of DNA.
Topoisomerases are
isomerase enzymes
that act on the
topology of DNA.

30. Roleof topoisomerase
 The winding problem of DNA arises due to the intertwined nature
of its double-helical structure.
 During DNA replication and transcription, DNA becomes
overwound ahead of a replication fork.
 If left unabated, this torsion would eventually stop the ability of
RNA & DNA polymerase involved in these processes to continue
down the DNA strand.
 Topoisomerases bind to either single-stranded or double-stranded
DNA and cut the phosphate backbone of the DNA.
 This intermediate break allows the DNA to be untangled or
unwound, and, at the end of these processes, the DNAbackbone
is resealed again.

31. Typesof topoisomerase
1)Topoisomerases I :
breaks only one strand
2)Topoisomerases II :
breaks both strands

32. TypeI topoisomerases
Type I topoisomerases are enzymes that cut one of the two
strands of double-stranded DNA, relax the strand, and reanneal the
strand.

33. TypeII topoisomerases
Type II topoisomerases cut both strands of the DNA helix
simultaneously. They use the hydrolysis of ATP, unlike Type I
topoisomerase. These enzymes change the linking number of
circular DNA by ±2.

34. Functions:
 To remove DNA supercoils during
transcription and DNA replication,
 For strand breakage during recombination,
 For chromosome condensation, and
 To disentangle intertwined DNA during
mitosis

38. THANK YOU