Mapping of origins of replication

Mapping of origins of
replication
BY GOPAL M. KUMBHANI
M. SC BIOTECHNOLOGY (SEM-1)
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Contents:-
 Basics about replication
 Why we are doing mapping of origins of replication
 Methods for mapping origins
 References
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What is replication?
 It is a process which make a synthesis of new DNA
strands.
 So, the process, It means that it should start from
some site. So, There is a question about from where
DNA replication is start?
 The answer is ORIGINS OF REPLICATION
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 It is a particular sequence in a genome from which
replication is initiated
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 Mostly replication is performed in bidirectional. In rare no. of
prokaryotes have unidirectional type of replication.
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 The genome size of the eukaryotes is very large. So, if they
want to make fast replication at that time bidirectional
replication is important.
 Also eukaryotes have multiple origins of replication because
of this reason.
 Replication start at an origin by separating the two strands of
DNA duplex.
 Each of the parent strand then act as template to synthesize a
complementary daughter strand.

 This model of replication in which a parental duplex give rise
to two daughter duplexes each containing one original
parental strand & one new strand is called semiconservative
mode of replication.
 The point at which replication occur is called replication fork or
also called as growing point.
 A replication fork moves from the starting point which is known
as origin.
 In bidirectional replication, two replication forks are formed.
They proceed away from the origin in opposite directions.
 In unidirectional replication, one replication fork leaves the
origin & proceed forward along the DNA.
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Why we do mapping of origins of
replication?
 Scientists had information about DNA replication but they
didn’t know about the site from where replication is start.
 So, they started mapping of origins of replication to find the
location of origin in the whole genome.
 Scientists first found replication origins in brewer’s
yeast(saccharomyces cerevisiae)
 Using traditional molecular approaches, scientists found only
about 10% of the origins(30 of about 400) predicted to
function in the yeast genome.
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 In prokaryotic cells, there is only one point of origin
and replication is occurs in two opposing directions at
the same time, and takes place in the cell cytoplasm.
 In Eukaryotic cells on the other hand, have multiple
points of origin, and use unidirectional replication
within the nucleus of the cell.
 In prokaryotes one or two origins are present but in
eukaryotic cell multiple origins are present.
 So, mapping of eukaryotic origins is harder than the
prokaryotic cells.
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Mapping of origins of replication can be done by
Autoradiography & 2-D Gel Electrophoresis
 There are many authore methods are available to
identify origins .
1)Electrophoresis
2)Microarray
3)Single-Standard DNA detection
4)DNA-Protein interaction at initial stage
5)Autoradiography
6)Electron microscopy
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 The choice of method depends upon the whether the DNA is a
defined molecule or undefined region of a cellular genome.
1) With a defined linear molecule,
 we can use electron microscopy to measure the distance of
each end of the bubble from the end of the DNA that have
bubbles of different sizes.
 If replication is unidirectional, only one of the end will move.
The other is fixed origin. So, It is easy to find the location of
origin in unidirectional replication in defined molecule.
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 If replication is bidirectional, both end will move; the origin is
the point midway between them.
2) With undefined regions of large genomes,
 Two successive pulse of labeled nucleotides can detect DNA
replication.
 Traditionally this was performed with radioactive DNA
precursors.
 However, recent advances in fluorescence labeling methods
have been made.
 Due to its simplicity this approach is become a system of
choice.
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 This shows the pattern of bidirectional replication generated
by initial labeling of DNA resulting in green DNA; the addition
of a second fluorescent label generates yellow DNA.
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2-D Gel electrophoresis For Mapping
Origins
 In 1987, Brewer and Fangman introduced the use of two-
dimensional agarose gels (2-D gels) to map origins of
replication.
 This technique was used to show that ARSs(autonomously
replicating sequences) act as initiation sites for DNA replication
in living cells.
 DNA isolated from rapidly proliferating cells is digested with a
restriction endonuclease.
 The fragment produced are resolved by neutral 2-D gel
electrophoresis.
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 In this technique restriction fragments of replicating DNA are
electrophored in a 1st dimension that separates by size & 2nd
dimension (run perpendicular to the first) that separates by the
shape.
 Once electrophoresis is complete, the DNA molecules are
transferred to nitrocellulose & detect by southern blotting.
 The most unusual structures migrate slowly in the first
dimension.
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 Molecules that contains an origin of replication form bubble
shaped replication intermediate that migrate even more slowly
in the 1st dimension than Y shaped molecules in which no
origin is present.
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 molecules that have just initiated replication are smaller and
will move fast in the first dimension. They will also have a
small replication bubble, and hence they will move fast in the
second dimension.
 However, those with more extensive replication will have a
larger bubble. These molecules are larger, and thus move
more slowly in the first dimension, but importantly, the larger
bubbles will move even more slowly in the second dimention,
since they have the greater deviation from linearity. This
generates a characteristic "bubble arc" on the two-
dimensional gel.
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 When a replication fork is passively generated from an origin
of replication that is outside the fragment, the Y-shaped
replication intermediates generate a Y-arc
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REFRENCES
 Lewin’s GENES X – Krebs, Goldstein, Kilpatrick [Page
no:265-268]
 Molecular biology of the gene(5th edition) – Watson, Levine,
Losick [Page no:214-216]
 Research article on method for mapping DNA replication
origins by Loretta D. Spotila and Joel A. Huberman
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