DNA REPLICATION (SEMICONSERVATIVE /ONION PEEL MODELS):
SEMICONSERVATIVE MODEL:
Semi-conservative replication is the mechanism by which DNA replicates in cells. The parent
strand splits in two and uses itself as a template to form a second complementary strand. The
exposed bases on the single-stranded DNA complementary base pair to nucleotides. A (Adenine)
pairs with T (Thymine) and C (Cytosine) pairs with G (Guanine). Together the template strand
and the complementary strand bond together to form a new double strand of DNA. One parent
double strand of DNA will thus become two daughter double strands of DNA .
The new strand of DNA is made in the 5' to 3' direction as a deoxyribonucleotide is added to the
3' OH end of the chain which is catalysed by DNA polymerase.
The term "semi-conservative" refers to the fact that each of daughter double helix contains one
conserved strand from the parent DNA, as well as one newly synthesised strand .
DNA REPAIR MECHANISM:
There are three major DNA repairing mechanisms: base excision, nucleotide
excision and mismatch repair.
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.
Figure 7-G-1. DNA repair by base excision.
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. In yeast, the proteins similar to Uvr's are named RADxx ("RAD" stands for
"radiation"), such as RAD3, RAD10. etc.
Figure 7-G-2. DNA repair by nucleotide excision.
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.
Figure 7-G-3. Mismatch repair.
The repairing process begins with the protein MutS which binds to mismatched base pairs.
Then, MutL is recruited to the complex and activates MutH which binds to GATC sequences.
Activation of MutH cleaves the unmethylated strand at the GATC site. Subsequently, the
segment from the cleavage site to the mismatch is removed by exonuclease (with assistance
from helicase II and SSB proteins). 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). If the cleavage occurs on the 5' side of the mismatch, exonuclease VII or RecJ is
used to degrade the single stranded DNA. The gap is filled by DNA polymerase III and DNA
ligase.