Replication Forks and Origins of Replication

In the cell, both strands of the DNA duplex are replicated at the same time. The first step in DNA replication is the separation of the two DNA strands that make up the helix that is to be copied to make two template DNAs. DNA Helicase untwists the helix at locations called replication origins. The junction between the newly separated template strands and the unreplicated duplex DNA is known as the replication fork.

The replication fork moves down the DNA strand, usually from an internal location to the strand’s end (unreplicated DNA). The result is that every replication fork has a twin replication fork, moving in the opposite direction from that same internal location to the strand’s opposite end.

Single-stranded binding proteins (SSB) work with helicase to keep the parental DNA helix unwound. It works by coating to stabilize the unwound strands with rigid subunits of SSB that keep the strands from snapping back together in a helix. Binding of one SSB promotes the binding of another SSB to the immediately adjacent ssDNA. This is called cooperative binding.The SSB subunits coat the single-strands of DNA in a way as not to cover the bases, allowing the DNA to remain available for base-pairing with the newly synthesized daughter strands.

As the strands of DNA are seperated at the replication fork, the duplex DNA infront of the fork becomes increasingly positively supercoiled. This accumulation of super is the result of DNA helicase eliminating the base parts between the two strands. If there is no mechanism to relieve the accumulation of these supercoils, the replicating machinery would grind to a halt in the face of mounting pressure. These super coils produced by the DNA unwinding at the replication fork is removed by Toposiomerases.

Because of the antiparallel nature of DNA and because DNA is only sythesized by elongating 3′ end, daughter strands synthesize through different methods, one adding nucleotides one by one in the direction of the replication fork, the other able to add nucleotides only in chunks. The first strand, which replicates nucleotides one by one (continuously) is called the leading strand; the other strand, which replicates in chunks (discontinuous fashion), is called the lagging strand.

Leading strand DNA polymerase can replicate its template as soon as it is exposed, synthesis of the lagging strand must wait for movement of the replication fork to expose a substantial length of new lagging strand is exposed.

The lagging strand replicates in small segments, called Okazaki fragments. These fragments are stretches of 100 to 200 nucleotides in humans (1000 to 2000 in bacteria) that are synthesized in the 5′ to 3′ direction away from the replication fork. Yet while each individual segment is replicated away from the replication fork, each subsequent Okazaki fragment is replicated more closely to the receding replication fork than the fragment before. These fragments are then stitched together by DNA ligase, creating a continuous strand. This type of replication is called discontinuous.

The initiation of New strand of DNA requires an RNA Primer with free 3’OH. DNA polymerase cannot initiate a new DNA strand them selves. To accomplish this the cell take advantage of the ability of RNA polymerase to do what DNA polymerases cannot: initiate new RNA strand. Primase is a special RNA polymerase dedicated to making short, RNA primers on an ssDNA template. These primers are subsequently extended by DNA Polymerase.

Although both the leading and lagging strands require primase to initiate DNA synthesis, the frequency of primase function on the two strands is dramatically different. Leading strand requires only a single RNA primer. In contrast, the discontinuous synthesis of the lagging strand means that new primers are needed for each Okazaki fragments.

Primase does not require a specific DNA sequence to initiate the synthesize of new RNA primer. Instead primase is activated only when it is associated with other DNA replication protein such as DNA helicase. Once activated, the primase synthesize a RNA primer using the most recently exposed lagging strand template regardless of sequence.

To complete the DNA replication, the RNA primase used for the initiation must be removed and replaced by DNA. to replace the RNA primers with DNA, an enzyme called RNase H recognize and removes most of the each RNA primer. This enzyme specifically degrades RNA that is base paired with DNA. RNase H removes all of the RNA primer except the ribonucleotide directly linked to the DNA end. This is because RNase H can only cleave bonds between tow ribonucleotides.The final ribonuceotide is removed by an a exonuclease, that degrages RNA and DNA from there 5′ end.

Removal of RNA leaves a gap in the double-stranded DNA. DNA polymerase fill the gap until every nucleotide is base-paired, leaving a DNA molecule that is complete except for the break in the backbone bvetween ythe 3’Oh and 5′ phosphate of the repaired strand. This nick in the DNA can be repaired by an enzymes called DNA ligase. Only after all RNA primase are replaced and asscociated nicks are sealed, DNA synthesis is completed.

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