TEKNOLOGI GEN

( EKSPRESSI GEN DAN DNA

REPAIR )

BLOK VII

CELL GROWTH
 DNA repair is
Involved in processes that minimize cell
killling, mutations, replication errors,
persistence of DNA damage and genomic
instability.

Abnormalities in these processes have

been implicated in cancer and aging
 Mutations may be caused

1. Spontaneous cellular processes

(replication errors, accidental
deamination, etc.)

2. External factors (UV light, chemical

agents, etc.)
 Replication errors
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.
a cell division requires synthesis of 6 X 10 9
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
 There are three major DNA
repairing mechanisms:

base excision
nucleotide excision
mismatch repair
 1. 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.
 Deamination( Fig.1)
The following figure illustrates the
deamination process. Conversion of the
methylated cytosine (methylcytosine) to
thymine may explain the observed CpG
islands
Fig1. Examples of deamination which involves the removal
of an amino group. Accidental deamination may change the
cytosine to uracil, or the methylated cytosine to thymine.
 CpG islands
The CG island is a short stretch of DNA in which the
frequency of the CG sequence is higher than other regions.
It is also called the CpG island, where "p" simply indicates
that "C" and "G" are connected by a phosphodiester bond.

CpG islands are often located around the promoters of

housekeeping genes (which are essential for general cell
functions) or other genes frequently expressed in a cell. At
these locations, the CG sequence is not methylated. By
contrast, the CG sequences in inactive genes are usually
methylated to suppress their expression.

the cytosine to uracil mutation which is efficiently repaired,

the cytosine to thymine mutation can be corrected only by
the mismatch repair which is very inefficient.
 Thus, during cell division, the
methylation pattern should also
pass over to the daughter cell.
This is achieved by the enzyme,
DNA methyltransferase, which
can methylate only the CG
sequence paired with
methylated CG.

Inheritance of the DNA methylation pattern. The DNA methyltransferase can methylate
only the CG sequence paired with methylated CG. The CG sequence not paired with
methylated CG will not be methylated.
1. Cytosine is deaminated forming uracil, the U can be
recognized as being an improper base in DNA by the
enzyme, uracil DNA glycosylase.
2. This enzyme cleaves the uracil base from the
phosphodiester backbone
3. the space is opened up by repair nucleases that
remove a number of nucleotides from one strand
4.The repair polymerase, DNA polymerase beta, then
fills in the gap and DNA ligase seals the last
phosphodiester bond.
5.The double strandedness of DNA makes possible both
DNA replication and DNA repair, because the template
strand always contains the information for the synthesis
of a complementary strand.
Base excision repair pathway (BER)
(a)A DNA glycosylase recognizes a damaged base and
cleaves between the base and deoxyribose in the
backbone.
(b) An AP endonuclease cleaves the phosphodiester
backbone near the AP site.
(c) DNA polymerase I initiates repair synthesis from the
free 3’ OH at the nick, removing a portion of the
damaged strand (with its 5’→3’ exonuclease activity) and
replacing it with undamaged DNA.
(d)The nick remaining after DNA polymerase I has
dissociated is sealed by DNA ligase.

AP= apurinic or apyrimidinic

(a=without)
5’ phosphate

O P O
Base
O

CH2
O

Sugar

O P O
Base
O

CH2 O
Sugar

3’ hydroxyl
OH
 2. Nucleotide excision
In E. coli, proteins UvrA, UvrB, and UvrC
are involved in removing the damaged
nucleotides (e.g., the dimer induced by UV
light). The gap is then filled by DNA
polymerase I and DNA ligase.
Photoreactivation
Nucleotide excision repair occurs
when the DNA lesion is larger,
for example when there
is a thymine dimer.
In this case,
a special repair excinuclease
removes about 30 nucleotides,
including the lesion.
The DNA is then resynthesized
and ligated together as with
base excision repair.
 Mismatch repair
To repair mismatched bases, the system has to
know which base is the correct one. In E. coli,
this is achieved by a special methylase called
the "Dam methylase", which can methylate all
adenines that occur within (5')GATC sequences.
Immediately after DNA replication, the template
strand has been methylated, but the newly
synthesized strand is not methylated yet. Thus,
the template strand and the new strand can be
distinguished.
1. The repairing process begins with the protein MutS which binds
to mismatched base pairs.
2. Then, MutL is recruited to the complex and activates MutH
which binds to GATC sequences.
3. Activation of MutH cleaves the unmethylated strand at the
GATC site.
4. Subsequently, the segment from the cleavage site to the
mismatch is removed by exonuclease (with assistance from
helicase II and SSB proteins).
5. If the cleavage occurs on the 3' side of the mismatch, this step
is carried out by exonuclease I (which degrades a single
strand only in the 3' to 5' direction).
6. If the cleavage occurs on the 5' side of the mismatch,
exonuclease VII or RecJ is used to degrade the single
stranded DNA.
7. The gap is filled by DNA polymerase III and DNA ligase.
 The distance between the GATC site and the mismatch could
be as long as 1,000 base pairs. Therefore, mismatch repair is
very expensive and inefficient.

 Mismatch repair in eukaryotes may be similar to that in E. coli.

Homologs of MutS and MutL have been identified in yeast,
mammals, and other eukaryotes.

 MSH1 to MSH5 are homologous to MutS;

 MLH1, PMS1 and PMS2 are homologous to MutL.

 Mutations of MSH2, PMS1 and PMS2 are related to colon

cancer.

 In eukaryotes, the mechanism to distinguish the template strand

from the new strand is still unclear.
 The main DNA polymerases required for DNA replication in E. coli
are DNA polymerases I and III. They both have 5' to 3'
polymerizing activity and 3' to 5' proofreading activity. DNA
polymerase I also removes the RNA primer and is also a DNA
repair enzyme, and thus requires a 5' to 3' exonuclease activity.
Mutasi yang terjadi selama replikasi DNA akan dapat diperbaiki selama memungkinkan
prooreading ( memperbaiki cetakan ) oleh DNA polymerase

Mutasi yang tidak dapat diperbaiki dengan proofreading akan diperbaiki dengan
mismatchhed repair ( post replication ) kemudian dengan excision repair

Mutasi yang terjadi secara spontan akan diperbaiki melalui excission repair ( base
excission atau nucleotide excission )