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>ZOOHCC - 501: Molecular Biology (Theory)
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> >Unit 2: DNA Replication > >
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> >DNA Replication > >
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>Prokaryotes have been the subject of extensive research on DNA > > >
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replication, largely because of their tiny genome sizes and the >
>availability of mutations. A single circular chromosome in E. coli >
>contains 4.6 million base pairs, and all of it replicates in roughly 42 >
>minutes, starting at a single origin of replication and moving in both >
>the circle. This indicates that almost a thousand >
>nucleotides are inserted every second. The procedure moves along rather >
>quickly and without many errors. >
>Proteins and enzymes are used in great quantity during DNA replication, >
>and each one is to the process. The enzyme DNA polymerase, >
>also known as DNA pol, which adds nucleotides that are complementary to >
>the template strand one at a time to the expanding DNA chain, is one of >
>the main participants. Energy is needed to add nucleotides, and it comes >
>from the nucleotides with three phosphates connected to them, much like >
>ATP has three phosphate groups attached. The energy produced when the >
>bond between the phosphates is broken is utilised to create the >
>bond between the new nucleotide and the expanding chain. >
>Three different forms of polymerases are known to exist in prokaryotes: >
>DNA pol I, DNA pol II, and DNA pol III. > >
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> >How does the replication machinery know where to begin? > >
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>It turns out that there are specific nucleotide sequences called >
>origins of replication where replication begins. In >
>E. coli, >
>which has a single origin of replication on its one chromosome (as do >most prokaryotes), it is approximately 245 base pairs long and is rich
>in AT sequences. The origin of replication is recognized by certain > >proteins that bind to this site. An enzyme called helicase unwinds the > >DNA by breaking the hydrogen bonds between the nitrogenous base
pairs. > >ATP hydrolysis is required for this process. As the DNA opens up, > >Y-shaped structures called replication forks are formed. Two > >replication forks are formed at the origin of replication and these > >get extended bi- directionally as replication proceeds.
Single-strand > >binding proteins coat the single strands of DNA near the replication > >fork to prevent the single-stranded DNA from winding back into a > >double helix. DNA polymerase is able to add nucleotides only in the 5' > >to 3' direction (a new DNA strand can be only extended in this > >direction). It also requires a free 3'-OH group to which it can add > >nucleotides by forming a phosphodiester bond between the 3'-OH end and > >the 5' phosphate of the next nucleotide. This essentially means that > >it cannot add nucleotides if a free 3'-OH group is not available. Then > >how does it add the first nucleotide? The problem is solved with the > >help of a primer that provides the free 3'-OH end. Another enzyme, RNA > >primase, synthesizes an RNA primer that is about five to ten > >nucleotides long and complementary to the DNA. Because this sequence > >primes the DNA synthesis, it is appropriately called the primer. DNA > >polymerase can now extend this RNA primer, adding nucleotides one by > >one that are complementary to the template strand >
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replication fork, DNA polymerase can only stretch in the 5' to 3'
direction, which causes a small issue. The DNA double helix is
anti-parallel, which means that one of its strands is orientated in the 5'
to 3' direction and the other in the 3' to 5' direction. Because the
polymerase may add nucleotides in this direction, one strand, which is
complementary to the 3' to 5' parental DNA strand, is continually created
in the direction of the replication fork. The leading strand is this one
that is always being created. Small pieces of the other strand, which is
complementary to the 5' to 3' parental DNA, are stretched away from the
replication fork. >
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>The lagging strand requires a new primer for each of the brief Okazaki
fragments, but the leading strand can be expanded by a single primer. The
lagging strand will be oriented generally in a 3' to 5' to 5' to 3'
orientation. As the DNA polymerase continues to add nucleotides, a protein
known as the sliding clamp keeps the enzyme in place. A ring-shaped
protein called the sliding clamp attaches to DNA and maintains the
polymerase in place. Topoisomerase works by temporarily nicking the DNA
helix and then resealing it to stop the DNA double helix from overwinding
as the DNA is opening up to the replication fork. The RNA primers are
changed as the synthesis progresses. >
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