REPLIKASI DNA

REPAIR DNA
REKOMBINASI DNA

Dr. Upik A. Miskad, PhD

Unhas Research Center
 An organism may contain
many types of somatic
cells, each with distinct
shape and
function. However, they
all have the same
genome. The genes in a
genome do not have any
effect on cellular
functions until they are
"expressed". Different
types of cells express
different sets of genes,
thereby exhibiting
various shapes and
functions.
 MEOSIS
• Meiosis is a special
type of cell division
resulting in four
genetically
nonequivalent
daughter cells, each
containing half the
number of
chromosomes of the
parent cell. Germ
cells (eggs and
sperm) are produced
by meiosis
 MITOSIS
• Mitosis is a process of
cell division resulting in
two genetically
equivalent daughter
cells. The period
between mitoses is called
the interphase which
covers the G1, S, and G2
phases of the cell
cycle. During the G2
phase, DNA has been
replicated and the
chromosome number is
4n. In the interphase,
chromosomes are not
visible by light
microscopy because the
chromatin diffuses
throughout the nucleus
 Chromosome and Gene
• Chromosome: pembawa informasi genetik
turunan yang dapat dilihat pada inti sel
ketika sel mulai membelah dalam bentuk
benang benang.
• Didalam kromosom terdapat gen-gen
yang dibentuk dari molekul-molekul DNA
dan terletak dalam suatu lokus.
• Ketika sel mulai membelah gen akan
membuat copy dari gen itu sendiri,
selanjutnya gen gen itu akan diturunkan
ke sel sel turunan.
 GENE

By definition, a gene includes the entire nucleic acid sequence

necessary for the expression of its product (peptide or RNA).
Such sequence may be divided into regulatory region and
transcriptional region.
 DNA
 DNA adalah suatu
polimer yang panjang
tidak bercabang,
mengandung 2 rantai
polynucleotida yang
tersusun sebagai suatu
bentuk antiparalel yang
mengelilingi aksis.
DNA
 REPLIKASI DNA

 Replikasi DNA adalah proses perbanyakan atau duplikasi

DNA
 Setiap DNA terdiri dari 2rantai polinucleotida dan
merupakan komplemen/ antiparalel.
 Setiap rantai bisa menjadi “TEMPLATE”/ cetakan untuk
pembentukan rantai partnernya
 Enzym yang diperlukan dalam proses replikasi adalah
 DNA polymerase,
 DNA primase, and
 cyclin
 DNA POLYMERASE
 Three types of DNA polymerases exist in E. coli:
I, II and III.
 The DNA polymerase I is used to fill the gap between
DNA fragments of the lagging strand. It is also the
major enzyme for gap filling during DNA repair.
 The DNA polymerase II is encoded by the PolB gene,
which is involved in the SOS response to DNA
damage.
 DNA replication is mainly carried out by the DNA
polymerase III.
 REPLIKASI DNA

DNA replication is triggered by the expression of all required

proteins, such as DNA polymerase, DNA primase, and cyclin
 Schematic drawing of the DNA
replication process.
• DNA replication starts from the
replication origin, proceeding
along both directions
• O1, O2, and O3 are replication
origins, each serving a region
called replicon (R1, R2, and
R3).
• DNA replication involves
unwinding of the double helix.
• New strands are synthesized by
DNA polymerases using the old
strands as template.
• Unwinding of a DNA molecule
looks like a "fork" growing in
one direction.
• The region being replicated
looks like a bubble called the
"replication bubble" (in red).
 Cara Syntesis DNA
• Penambahan satu DNA yang telah terfosphorilasi
ke 3’ end dari polinucleotida dengan bantuan
enzim polimerase/primase
• Diawali degan pemisahan lokal ke 2 rantai DNA
yang saling berkomplemen.
• Setiap rantai bertindak sebagai template.
• Akhirnya template membentuk satu rantai DNA
yang baru yang merupakan komplemennya
• Karena setiap turunan DNA mengandung 1 rantai
DNA induk (template) dan satu rantai DNA
replikasi baru maka proses ini disebut replikasi
semikonservatif
 DNA REPLIKASI
• DNA molecules are synthesized by DNA
polymerases from deoxyribonucleoside
triphosphate (dNTP).
• The chemical reaction is similar to the synthesis
of RNA strands.
• Both DNA and RNA polymerases can extend
nucleic acid strands only in the 5' to 3'
direction. However, the two strands in a DNA
molecule are antiparallel.
• Therefore, only one strand (leading strand) can
be synthesized continuously by the DNA
polymerase. The other strand (lagging strand) is
synthesized segment by segment.
 Synthesis DNA
• There is a major difference between DNA polymerase and
RNA polymerase: the RNA polymerase can synthesize a
new strand whereas the DNA polymerase can only extend
an existing strand.
• Therefore, to synthesize a DNA molecule, a short RNA
molecule (~ 5 - 12 nucleotides) should be synthesize first
by a specific enzyme. The initiating RNA molecule is known
as the primer, and the enzyme is called primase.
• In addition to DNA polymerase and primase, DNA
replication requires helicase and single strand binding
protein (SSB protein). The role of helicase is to unwind
the duplex DNA.
• SSB proteins can bind to both separated strands,
preventing them from annealing (reconstitution of double-
stranded DNA from single strands).
 Synthesis DNA
• DNA polymerases can extend nucleic acid
strands only in the 5' to 3'
direction. However, in the direction of a
growing fork, only one strand is from 5' to
3'.
• This strand (the leading strand) can be
synthesized continuously. The other
strand (the lagging strand), whose 5' to 3'
direction is opposite to the movement of a
growing fork, should be synthesized
discontinuously.
•
Steps in the synthesis of the
lagging strand.
• (a) Comparison between the leading strand and the
lagging strand.
• (b) The primase first synthesizes a new primer which is
about 10 nucleotides in length. The distance between two
primers is about 1000-2000 nucleotides in bacteria, and
about 100-200 nucleotides in eukaryotic cells.
• (c) DNA polymerase elongates the new primer in the 5' to
3' direction until it reaches the 5' end of a neighboring
primer. The newly synthesized DNA is called an Okazaki
fragment.
• (d) In E. coli, DNA polymerase I has the 5' to 3'
exonuclease activity, which is used to remove a primer.
• (e) DNA ligase joins adjacent Okazaki fragments.
• The whole lagging strand is synthesized by repeating steps
(b) to (e).
 DNA DAMAGE
• DNA Damage is caused by a variety
of agents and by different
mechanisms
• Replication errors may cause
mismatched bases
• Cells of all types have a number of
DNA repair pathways to deal with
mismatches and damage
 Cause of DNA Damage
• Irradiation
• Chemical
• Note: Mutagens change the DNA
sequence but not necessarily by
DNA damage (mostly chemical
change from one base to another
base)
 X-ray Irradiation

• X rays break the DNA backbone

Oxidation from free radicals
(formed by irradiation)
damages individual bases
 MUTATION
• Mutasi adalah perubahan yang terjadi pada rangkain DNA
yang menyebabkan kesalahan genetik.
• Mutasi dapat menyebabkan perubahan protein yang dikode
oleh gen tersebut atau tidak dikodenya pembentukan
protein.
 MUTATION
• Mutation refers to the change in a DNA
sequence, which may involve only a few
bases or the large-scale chromosome
abnormality.

• This section covers the small-scale

mutations (substitution, deletion,
insertion) and the exon skipping that
results from mutation at the splice site.
Deamination
Depurination
 SUBTITUTION
In the substitution mutation, one or more
nucleotides are substituted by the same number
of different nucleotides.
In most cases, only one nucleotide is changed.

Based on the change in the nucleotide type, the

substitution mutation may be divided into
transition and transversion mutations.

Based on the consequence of mutation, the

substitution mutation may be grouped into
silent, missense and nonsense mutations.
 SUBTITUTION
The substitution mutation.
(a) Illustration of transition
(blue) and transversion (red)
mutations. In the transition
mutation, a pyrimidine (C or T)
is substituted by another
pyrimidine, or a purine (A or G)
is substituted by another
purine. The transversion
mutation involves the change
from a pyrimidine to a purine, or
vice versa.

(b) Examples of silent, missense

and nonsense mutations. The
silent mutation does not
produce any change in the
amino acid sequence, the
missense mutation results in a
different amino acid, and the
nonsense mutation generates a
stop signal.
 INSERTION
• In the insertion mutation, one or
more nucleotides are inserted into a
sequence.
• If the number of inserted bases is
not a multiple of 3, it will cause
frameshift, resulting in serious
consequences.
•
 INSERTION
• As shown in the following table, non-frameshift
insertions may also cause diseases.
 DELETION
The deletion mutation involves
elimination of one or more
nucleotides from a DNA sequence. It
may cause frameshift, producing a
non-functional protein
 DELETION

Real examples of deletion mutations which cause diseases. (a) Deletion of "T" from
the sequence "TTTTT" in the CFTR gene.
(b) Deletion of "AT" from the sequence "ATAT" in the CFTR gene.
(c) Deletion of "TTG" from the sequence "TTGTTG" in the FIX gene.
(d) Deletion of "ATAG" from the sequence "ATAGATAG" in the APC gene.
Note that deletion and insertion mutations often occur in the repetitive sequence. As
explained in the next section, they are usually caused by "replication slippage".
 REPLICATION ERROR
• Replication errors are the main source of mutations.
• It has been estimated that uncorrected replication errors
occur with a frequency of 10-9 - 10-11 for each nucleotide
added by DNA polymerases.
• Since a cell division requires synthesis of 6 X 109
nucleotides, the mutation rate is about one per cell
division.
• A commonly observed replication error is the replication
slippage, which occurs at the repetitive sequences when
the new strand mispairs with the template strand.
• The microsatellite polymorphism is mainly caused by the
replication slippage.
• If the mutation occurs in a coding region, it could produce
abnormal proteins, leading to diseases. The Huntington's
disease is a well known example
Mutation by Replication Errors

• The mutation caused by

replication slippage. In
this figure, mispairing
involves only one
repeat. In fact, the
slippage could cause
several repeats to
become unpaired.
• (a) Normal replication.
• (b) Backward slippage,
resulting in the insertion
mutation.
• (c) Forward slippage,
resulting in the deletion
mutation.
 DNA REPAIR
• There are three major DNA repairing
mechanisms:

• base excision,
• nucleotide excision and
• mismatch repair.
 DNA REPAIR

• Proteins involved in the DNA repairing of

E. coli.
BASE EXCISION
• DNA's bases may be
modified by deamination
or alkylation.
• The position of the
modified (damaged) base
is called the "abasic site"
or "AP site".
• In E.coli, the DNA
glycosylase can recognize
the AP site and remove
its base.
• Then, the AP
endonuclease removes
the AP site and
neighboring nucleotides.
• The gap is filled by DNA
polymerase I and DNA
ligase.
 Nucleotide excision

• In E. coli, proteins
UvrA, UvrB, and
UvrC are involved in
removing the
damaged
nucleotides.
• The gap is then filled
by DNA polymerase I
and DNA ligase.
 DNA RECOMBINATION
• DNA recombination refers to the process that a
DNA segment moves from one DNA molecule to
another DNA molecule. The following three types
are most commonly observed.

• Homologous recombination
• Site-specific recombination
• Transpositional recombination

• The homologous recombination often occurs

during meiosis. Other types of recombination are
not specifically related to cell division.
• Comparison between gene conversion and DNA crossover.
• (a) Two DNA molecules.
• (b) Gene conversion - the red DNA donates part of its
genetic information (e-e' region) to the blue DNA.
• (c) DNA crossover - the two DNAs exchange part of their
genetic information (f-f' and F-F').
•Thank you and Good Luck