Saturday, June 28, 2008

DNA Replication

Origin of replication
For a cell to divide, it must first replicate its DNA. It occurs at S phase of the cell cycle. This process is initiated at particular points within the DNA, known as origin of replication or ‘Ori’. In a bacterium or virus DNA has only one origin of replication. Eukaryotic DNA is a giant molecule so they have number of origins of replication because of its large size and association with proteins. Origins contain DNA sequences recognized by replication initiator proteins (e.g. DNAA in E coli' and the Origin Recognition Complex in yeast).
Initiator proteins recruit other proteins to separate the DNA strands at the origin, forming a bubble. Origins tend to be "AT-rich" (rich in adenine and thymine bases) to assist this process because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair)—strands rich in these nucleotides are generally easier to separate.
Replication actually precedes bidirectional in both prokaryotes and eukaryotes except in coli phage P2 chromosome
Steps of Replication
1. Unwinding of DNA- The two strands of DNA separate. There are enzymes Helicases which unwind the DNA and separate the two strands. It involves breaking the weak Hydrogen bonds present in between the two strands of DNA. Now the two strands are free to act as template. Single stranded DNA binding proteins (SSBS) selectively bind to the single stranded DNA strands to stabilize this condition. Unwinding also creates a coiling of tension. This tension is released by topoisomerase. In prokaryotes topoisomerase and helicases are replaced by DNA gyrases.

2. The replication fork
Whole of DNA does not open in one stretch, due to very high energy requirement, the point of separation proceeds slowly from one end to another, it gives the appearance of Y shape structure called the replication fork
3. DNA strands serve as template. The enzyme DNA polymerase III in E. coli play an important role in adding the buildings blocks (nucleotides) it is a very fast process and the building blocks of DNA are not present in the form of deoxyribonucleotides but in the form of deoxyribonuclesides triphosphates. It is an energy consuming process. Deoxyribonuclesides triphosphates have two roles in DNA replication. It acts as a substrate; it also provides energy for polymerization reaction. As the two terminal phosphates are high energy molecules as in ATP.

4. The enzyme is active only in presence of Mg2+ and preexisting DNA.

5. DNA polymerase cannot initiate the synthesis of DNA. It needs RNA primer, which is a short stretch of RNA formed on the DNA template. The enzyme which polymerizes the RNA building blocks (AUCG) into the primer is known as primase, it contains free 3’OH.
6. DNA polymerase needs a free 3’OH on DNA. After start of nucleotide chain RNA primer is removed by a 5’→ 3’. Exonuclease enzyme.

Leading strand synthesis
The two separated DNA strand in the replication fork function as template. The DNA polymerase can polymerize the nucleotides only in the 5’ → 3’ direction.
Since the two strands of DNA are in ant parallel orientation (opposite direction), One is in 5’ → 3’ direction and the other is in 3'→5' direction. Replication of the two templates proceeds in two opposite direction. The synthesis is continuous in 3’→5' strand as its 3’ end is open for elongation. This strand is known as leading strand.

Lagging strand synthesis
The lagging strand is the DNA strand of replication fork running in the 3' to 5' direction. Because DNA polymerase cannot synthesize in the 3'→5' direction, the lagging strand is synthesized in short segments known as Okazaki fragments. Along the lagging strand's template, primase builds RNA primers in short bursts. DNA polymerases are then able to use the free 3' OH groups on the RNA primers to synthesize DNA in the 5'→3' direction. The RNA fragments are then removed (different mechanisms are used in eukaryotes and prokaryotes) and new deoxyribonucleotides are added to fill the gaps where the RNA was present by DNA polymerase I. DNA ligase then joins the deoxyribonucleotides together, completing the synthesis of the lagging strand.
Proof Reading and DNA repair
A wrong base is sometimes introduced during replication. The frequency is one in ten thousand. DNA polymerase I and III are able to sense the same. It goes back removes the wrong base and allows addition of proper base and then proceeds forward in the 5'→3' direction

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