Prominent important components of replication

1. Important components of
Replication Machinery
Dr. Asif Mir (Chairperson DBI&BT)

2. DNA helicases (require ATP)
• They are motor proteins that move directionally along a
nucleic acid phosphodiester backbone.
• The process of breaking the hydrogen bonds between the
nucleotide base pairs in double-stranded DNA
requires energy. To break the bonds, helicases use the
energy stored in a molecule called ATP, which serves as
the energy currency of cells.
• Approximately 1% of eukaryotic genes code for helicases.
•
• The human genome codes for 95 non-redundant
helicases: 64 RNA helicases and 31 DNA helicases.
• Many cellular processes, such as DNA replication,
transcription, translation, recombination, DNA repair,
and ribosome biogenesis involve the separation of nucleic
acid strands that necessitates the use of helicases.

3. Single stranded DNA binding
proteins (SSBs)
• Single-stranded DNA-binding proteins (SSB) have high
affinity to single-stranded (ss) DNA and participate in
DNA replication, recombination, and repair as accessory
protein .
• SSB plays a role in separating DNA strand during
replication and prevent ssDNA from re-form a double
helix.
• There are two kinds of complexes of SSB-ssDNA in
different site sizes in the (SSB)35- and (SSB)65- binding
modes.
• Single-stranded DNA can interact with two SSB subunits
in the (SSB)35 complex, which has "smooth-contoured"
structure as well as with all four SSB subunits in the
(SSB)65 complex, which has "beaded" structure.

4. DNA topoisomerase
• DNA topoisomerases are ubiquitous enzymes found in
all cell types from viruses to man.
• These enzymes act to regulate DNA supercoiling by
catalysing the winding and unwinding of DNA
strands.
• They do this by making an incision that breaks the
DNA backbone, so they can then pass the DNA
strands through one another, swivelling and
relaxing/coiling the DNA before resealing the breaks.

5. DNA topoisomerase I & II

6. DNA primase
• DNA primases are enzymes whose continual activity is
required at the DNA replication fork.
• They catalyze the synthesis of short RNA molecules
used as primers for DNA polymerases.
• Primers are synthesized from ribonucleoside
triphosphates and are four to fifteen nucleotides long.
• Most DNA primases can be divided into two classes.
The first class contains bacterial and bacteriophage
enzymes found associated with replicative DNA
helicases.
• The second major primase class comprises
heterodimeric eukaryotic primases that form a complex
with DNA polymerase alpha and its accessory B subunit.

7. DNA polymerase
• The structure of DNA polymerase is highly conserved, meaning
their catalytic subunits vary very little from one species to
another, irrespective of how their domains are structured.
• This highly conserved structure usually indicates that the
cellular functions they perform are crucial and irreplaceable
and therefore require rigid maintenance to ensure their
evolutionary advantage
• The DNA polymerases are enzymes that create DNA molecules
by assembling nucleotides, the building blocks of DNA. These
enzymes are essential to DNA replication and usually work in
pairs to create two identical DNA strands from one original
DNA molecule.
• During this process, DNA polymerase “reads” the existing DNA
strands to create two new strands that match the existing ones.

8. DNA ligase (require ATP)
• DNA ligases close nicks in the phosphodiester
backbone of DNA.
• Biologically, DNA ligases are essential for the joining
of Okazaki fragments during replication, and for
completing short-patch DNA synthesis occurring in
DNA repair process.
• The reaction occurs in three stages in all DNA ligases:
1. Formation of a covalent enzyme-AMP
intermediate linked to a lysine side-chain in the
enzyme.
2. Transfer of the AMP nucleotide to the 5’
phosphate of the nicked DNA strand.
3. Attack on the AMP-DNA bond by the 3’-OH of
the nicked DNA sealing the phosphate backbone and
resealing AMP.

9. DNA glycolyases
• DNA glycosylases remove lesions
generated by deamination of bases,
alkylating agents, oxidative stress, ionizing
radiation, or replication errors.
• All these lesions cause little perturbation
of DNA structure.
• Most DNA glycosylases excise a wide
variety of modified bases, while few of
them have, so far, a very narrow substrate
specificity.

10. Telomere
• The ends of the linear
chromosomes are known
as telomeres.
• Repetitive sequences that code
for no particular gene.
These telomeres protect the
important genes from being
deleted as cells divide and
as DNA strands shorten
during replication.