DNA REPLICATION: Before the lagging-strand DNA exits the replication factory, its RNA primers must be removed and the Okazaki fragments must be joined together to create a continuous DNA strand. The first step is the removal of the RNA primer. RNAse H, which recognizes RNA-DNA hybrid helices, degrades the RNA by hydrolyzing its phosphodiester bonds. Next, the sequence gap created by RNAse H is then filled in by DNA polymerase which extends the 3' end of the neighboring Okazaki fragment. Finally, the Okazaki fragments are joined together by DNA ligase that hooks together the 3' end of one fragment to the 5' phosphate group of the neighboring fragment in an ATP- or NAD+-dependent reaction. REPLICATION IN ACTION We are now ready to review the steps of DNA replication. The process begins when the helicase enzyme unwinds the double helix to expose two single DNA strands and create two replication forks. DNA replication takes place simultaneously at each fork. The mechanism of replication is identical at each fork. Remember that the proteins involved in replication are clustered together and anchored in the cell. Thus, the replication proteins do not travel down the length of the DNA. Instead, the DNA helix is fed through a stationary replication factory like film is fed through a projector. Single-strand binding proteins, or SSBs, coat the single DNA strands to prevent them from snapping back together. SSBs are easily displaced by DNA polymerase. The primase enzyme uses the original DNA sequence as a template to synthesize a short RNA primer. Primers are necessary because DNA polymerase can only extend a nucleotide chain, not start one. DNA polymerase begins to synthesize a new DNA strand by extending an RNA primer in the 5' to 3' direction. Each parental DNA strand is copied by one DNA polymerase. Remember, both template strands move through the replication factory in the same direction, and DNA polymerase can only synthesize DNA from the 5' end to the 3' end. Due to these two factors, one of the DNA strands must be made discontinuously in short pieces which are later joined together. As replication proceeds, RNAse H recognizes RNA primers bound to the DNA template and removes the primers by hydrolyzing the RNA. DNA polymerase can then fill in the gap left by RNase H. The DNA replication process is completed when the ligase enzyme joins the short DNA pieces together into one continuous strand.
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The first step in DNA replication is to 'unzip' the double helix structure of the DNA Molecule. This is carried out by an enzyme called helices which breaks the hydrogen bonds holding the complementary bases of DNA together [A with T, C with G]. The separation of the two single strands of DNA creates a 'Y' shape called a replication 'fork'. The two separated strands will
act as templates for making the new strands of DNA. One of the strands is oriented in the 3' to 5' direction [towards the replication fork], this is the leading strand. The other strand is oriented in the 5' to 3' direction [away from the replication fork], this is the lagging strand. As a result of their different orientations, the two strands are replicated differently:
Leading Strand: A short piece of RNA called a primer [produced by an enzyme called primase] comes along and binds to the
end of the leading strand. The primer acts as the starting point for DNA synthesis. DNA polymerase binds to the leading strand and then 'walks' along it, adding new complementary nucleotide bases [A, C, G and T] to the strand of DNA in the 5' to 3' direction. This sort of replication is called continuous.
Lagging strand: Numerous RNA primers are made by the primase enzyme and bind at various points along the lagging strand. Chunks of DNA, called Okazaki fragments, are then added to the
lagging strand also in the 5' to 3' direction.This type of replication is called discontinuous as the Okazaki fragments will need to be joined up later. Once all of the bases are matched up [A with T, C with G], an enzyme called exonuclease strips away the primer[s]. The gaps where the primer[s] were are then filled by yet more complementary nucleotides.The new strand is proofread to make sure there are no mistakes in the new DNA sequence.
Finally, an enzyme called DNA ligase? seals up the
sequence of DNA into two continuous double strands.The result of DNA replication is two DNA molecules consisting of one new and one old chain of nucleotides. This is why DNA replication is described as semi-conservative, half of the chain is part of the original DNA molecule, half is brand new. Following replication the new DNA automatically winds up into a double helix.