DEOXYRIBONUCLEIC ACID (DNA):
is the chief component of any living organism.it is responsible for the
transfer of genetic information from one generation to the next; this trait
makes DNA hereditary material. Monomer of DNA is
nucleotide that takes up sugar, phosphate group and a base from the four
Ø “A” pairs with “T” (A=T)
Ø “C” pairs with “G” (C=T)
Ø DNA exists in double helical form in
which strands of DNA are anti-parallel to each other.
Ø Errors in DNA strings are possible
& very frequent in case of prokaryotes & called DNA DAMAGE.
Ø This damage could be in the loss of
nucleotides or strand breakage etc.iii
type of genetic recombination in which exchange of nucleotide sequences occur
between two similar or identical molecules of DNA is called recombination”.
the exchange of genetic material in different species of bacteria and viruses
the process of recombination is used.
three main processes involved in recombination of bacteria:
process through which by the direct contact transfer of genetic material from
one bacterium to the other takes place contact is called conjugation.”
In E. coli this process was postulated
by Joshua Lederberg and Edward Tatum (1946).
In 1958, they were also awarded Nobel price
on the work of salmonella, vibrio and pseudomonas.
the process of conjugation there are two bacteria one donates the genetic
material and other act as recipient which receives the DNA.
factor or F factor is present in the donor cell. The F factor allows the
formation of pilus in the donor cell. With the help of pillus the donor cell
contact the recipient cell.
the pilus two bacteria come together and donor transfer genetic material to the
last step, both cells containing single stranded DNA, replicate it and form
double stranded F plasmid which is identical to the original one. Now both
cells are donors or F+ because F-plasmid has ability for the pili
genetic material is in the form of plasmid, or a small, circular DNA piece.
that carries antibiotic resistance genes can also transferred by the
“In molecular biology, transformation is the genetic modification of a cell by the direct uptake and expression
of DNA from its surroundings usually through the liquid medium.”
Ø It was described by FREDERICK GRIFFITH in 1928
are two types of transformation:
Artificial or forced transformation
type of transformation in which no force is required for the transfer of
genetic material is natural transformation.”
The transfer of isolated fragment is transformation
Ø The transformation takes place in the mixed population. The
break in the DNA of donor bacteria take apart and then the release and
fragmentation of isolated DNA take place.
Ø Then this fragmented DNA gets attached with the recipient
Ø During passage one strand dissolved by enzymes and single strand of
double stranded get attached with the recipient single stranded DNA.
incorporated DNA transformed new character in the bacteria.
and Rec A a special type of protein which is required in this process. It form
coating around the single stranded DNA and provide protection
the laboratory, by applying high voltage
electric field and by high concentration of CaCI2. Under such
condition, the bacterial cells are forced to take up foreign DNA. This type of
transformation is called artificial”.
this process recipient cells are able to take up double stranded DNA by the
physical and chemical processes.
A protein, mediated in the process of integration of foreign DNA with the
is another process of promoting competence. By this method, the cells are
shocked for the short time with an electric field of 10-20 kV/cm which will
create holes in the cell membrane for the entry of plasmid DNA. After the
electric shock, the holes are rapidly closed by the cell’s membrane-repair
cation in cold conditions also weakens the cell surface structure so that
transformation becomes easy.
process in which genetic material is exchanged from one living organism to
mechanism is discovered by two scientists namely Joshua Leaderberg & Nortor
mechanism occurs in bacteriophage
There are two types of
the bacterial cell wall, a receptor site is present.
this receptor site bacteriophage gets attached to the virus to which it attacks
and it transfers its nucleic Acid to the host cell.
transferred nucleic acid encodes specialized protein in host cells.
proteins may include repressor protein.
types of proteins inhibit the replication in bacteria by this repressor
viral chromosomes carry with it the part of bacteriophage nucleic acid &
virus with prophage step is called LYSOGENIC and the peaceful collaboration of
bacteriophage & virus is called LYSOGENY.
lysogenic phase continuous many generations and the time till both remains
together both types of genetic material transfers to the progeny.
this phage genetic material gets separate from the other and starts
synthesizing its own genetic material via replication & stats
kind of transduction is named as specialized transduction.
start the mechanism of both type of transduction is same.
only difference is that after bursting of host cell the bacteriophage contain
some fragments of the viral genome.
OF DNA DAMAGE:
There are numerous reasons to damage
to the DNA. Some of which are given below:
Ø DNA damage by oxidation
Ø Hydrolytic attack on DNA
Ø Free radicals from normal metabolic
Ø Errors in DNA synthesis
Ø Mutagens exposure
Ø Breakage of strands
Ø Loss of base
Ø Damage to sugar
Ø Damage of base
OF REPAIR MECHANISMS IN PROKARYOTES:
Following are some repair mechanisms
(a) Broken fork repair
(b) Double strand break repair
(c) Gap filling recombination repair
(a) Mismatch repair
(b) Base excision repair
(c) Nucleotide excision repair
Single strand breaks
Error prone repair
Double strand breaks
OF RECOMBINATION AND REPAIR MECHANISM
Recombination is called homologous
if the recombination occurs in identical type of chromosomes.
If we talk about mechanism of
repairing in prokaryotes here recombination reactions are used and three types
are used in repairing. The name of this useful mechanism is following:
Ø Broken fork repair
Ø Double-strand break repair
Ø Recombination gap-?lling repair
fork which is damaged in replication is getting back. This damaged fork is
subjected to restoration. After regaining its activity this replication fork is
able to perform its activity. On stolen template with the help of replication
these forks appear. Endonucleases may present this type of forks.
Mutation is the main
reason which arise this forks for replication. Nicked or damaged chromosome is
tarnished if recombination does not repair damaged forks.
3′ end is exposed, of the damaged chromosome by consuming the other strand.
displacement loops or D-loop is synthesized.
loop created by entering the unprotected 3′ end in to the duplex of homologous.
this way one strand from duplex is get displaced & an intermediate branch
shape is produced. Here branch migration occurs
by the movement of exposed end into the duplex and the outside movement of the
branch of duplex. Both of these move opposite to each other.
Holliday Junction is made by
holiday junction is basically a branch of four strands. Damaged fork activity
is regained by ligation of strands from D-loop & Holliday junction.
mechanism is detailed & extensively studied in prokaryote i.e. E. coli. In E. coli, this mechanism is also
having many names.
Dependent Replication” (RDR)
Strand End Repair (DSE repair)
these are the names of this mechanism.
repairing of the damaged fork is completed.
another important step, the arise of replisome occurs. Replication
is restarted by these replisome.in prokaryotes unique features are required to
repair have further types. All of which have same mechanism of following steps:
enzyme and molecules are used to cut the damaged part of DNA.
here DNA polymerase helps to remove the damage.
copies the damaged part. It helps in synthesis at 3’OH.
synthesis starts at site of nick or gap.
here ligases are used to join the DNA.
3 steps are involved in all three-excision repair mechanism namely
Miss-match repair (MMR)
Base-excision repair (BER)
Nucleotide Excision Repair (NER)
MISSMATCH REPAIR OR INSERTION-DELETION LOOP (MMR):
system is used for the repairing of bases which are wrongly inserted, or any
error related to the bases in DNA.
tautomerization is the main cause of miss-match.
is helpful in repairing, recombination & replication in case of any error.
specificity is one of the characteristic of this type of repair.
replication process, usually errors are usually present in daughter strand.so
for repair initiation the first step is to recognize the daughter strand.
hemi-methylation plays the main role in recognition of parental strand from
to DNA by different sources like internal or external could also be removed by
just recognition of the daughter or newly synthesized is done & then
wrongly inserted base or damage is repaired.
main work is to identification of daughter strand.in eukaryotes &
prokaryotes the recognition mechanism is different.
in gram-negative bacteria this mechanism is still known while in other
organisms it is still unknown.
G & T, C & G both type of pairing fall in the category of miss-match
By this method
thousands of repairs could be done.
EXCISION REPAIR (BER):
mechanism is also used in repairing the damage of DNA.
the whole cycle of cell this mechanism is useful.
helpful in removing the small lesions of damage.
repair differs from nucleotide excision repairs.
difference is in the recognition steps where substrate varies.
This system also removes that type of
damage even which is not involved in distortion of DNA.
Although G, U is not involved in the
distortion of DNA but still uridine is removed damage base.
The other difference removal of purines
or pyrimidines from DNA. This is done by the breakdown of glycosidic bond.
are used here. This helps to identify the damaged bases in DNA.
resolving the N-glycosidic bond glycosylases done their work.
Removal of defected bases is the
function of glycosylases.
Function of DNA polymerase is done by
the help of AP endonuclease by facilitating with primer.
This DNA polymerase removes bases &
fills these gaps via exonuclease & polymerase activity respectively.vii
basic principle of this mechanism is same like other excision mechanisms.
is used to remove the damage that is caused by UV-light & chemical adducts
mechanism occurs both in eukaryotes as well as prokaryotes.
prokaryotes, this system uses usually UvrABC exonuclease & UvrD helicase. viii
homologous recombination third type of repair is single-strand DNA gaps for
filling of this gap the reaction between homologous chromosomes occurs in which
gap due to incomplete replication can also be produced in daughter-strand. The
incomplete replication can occur due to damage in template strand, DNA
structure or protein that play role in binding.
of the first mechanisms for recombinational repair (Rupp et al., 1971) was gap
repairing in daughter strand (DSGR) it is the most poorly understood.
process has been best studied in E. coli after UV irradiation, where it has
been called “post-replication repair.”
daughter strands with gaps are having low molecular weight and that was
previously assayed by using thymidine labeling and denaturing sucrose gradient
centrifugation. By this, new strand become complemented with the parental
strand. This signifies the mechanism of Recombinational gap filling.
gap in the single-strand is complement with the intact DNA molecule.
strand invaded and invasion may be aided by displacement by one or both of the
strands that flank the break.
the synapsis does not involve a free end, to overcome this problem
topoisomerases are used in this process.
synthesis starts from the ends by replacing the transferred strand, setting up
a branched molecule, consisting of one or more crossed strands.
duplex molecules are restoring by cleavage and the gap is filled.
or non-crossover products are produced by the cleavage of these junctions, as
in DSB model.ix
DOUBLE-STRAND BREAK REPAIR:
Break in double-strand are
repaired by 2 different types of mechanism.
Interactions in homologues chromosomes
repair the breaks in the double strand.
E. coli lacking the non-homologous end
joining pathway, so homologous recombination is the only way to recover broken
Yeast mitotic recombination process can also
understand by DSB repair.
or Holliday junction is formed by the invasion of broken end; in this
intermediate displaced strand can recruit and pair with the second resected
information can be restored by DNA synthesis in paired intermediate which had
lost from any broken end.
Holliday junctions are created by this process (a feature noticeably confirmed
for yeast meiotic recombination intermediates)
intact chromosomes are produced by the resolution of Holliday junction, which
are “crossover” or “non-crossover” products (may or may not have exchanged the
the shape of E.coli chromosome is circular, dimeric circular chromosome will
form by crossing over between sisters chromosome.
XerCD is present at various sites near the terminus which is important in the
site-specific recombination system, this protein converted dimers into monomer
for appropriate separation to daughter cells.
an alternative version of double-strand break repair is also known as “synthesis-dependent strand annealing”
(SDSA), capture of the two
broken ends occurs sequentially.
synapsis of the second end is dependent on the successful synapsis of the first
end. After the invasion of first strand the DNA synthesis happens from the
invading strand which extends it away from the point of the original break.
the process ended the invading strands are separated from the intermediates for
the annealing of other processed broken ends.
process of break repair accomplishes, without the cleavage of any branched
structure, and so crossover products are not yielded.
E. coli, DSB repair has been studied by introducing double-strand breaks from
sequence-specific endonucleases and excision of transposable elements. x
CAUSES OF BREAKS:
Ionizing radiation causes:
strand break also produces DSBs
lesion to bacteria.
in sugar oxidation and base.
It is necessary to have intact
sister chromosome for the repairing of broken strand although homologous
sequence recombination with other chromosome can also be detected.xi
practice, it is difficult to distinguish between the one-ended replication fork
break repair and two-ended double-strand break repair.
both (double-stranded and replication fork) are not associated with each other.
But an agent that causes the DSB also produces broken forks.
also differ from each other in the aspect that repair of two ends correspond
each other, so it may require particular functions which is not required for
single-end break repair.
the other hand, uncoordinated and independent one-ended repair is involved in
repair of two broken end and with each process establishing a replication fork.
filled recombination differs from the DSB recombination process due to the
proteins are involved in this mechanism as compare to the DSB mechanism.
addition, due to incomplete replication single-strand gaps are produced which
will bind by single stranded DNA binding protein. In the complete process,
recombination protein will remove single stranded break.
mechanism of gap filling recombination was not studied earlier in E.coli. only
DSB-mediated processes were reported after conjugation and transduction.
DNA damage repair, it can be difficult to distinguish between DSB repairs and
gap filled recombination repair.
are also produced by the agents that produce gaps e.g., UV irradiation.
the process of DNA damage repair, gap repair are usually underestimated and
that gaps which are not repaired converted to the DSB and which will further
repaired by the DSB repair mechanism.
Ø This mechanism occurs occasionally in
Ø Especially in prokaryotes this mechanism does
Ø While some prokaryotes also still use this
mechanism of homologous end joining.
Ø Here in prokaryotes just in little number of
proteins all mechanism of non-homologous end joining is done.
Ø Here this mechanism occurs in its simplest