Despite differences in the nature of bacterial and eukaryotic genomes, the basic process of genomic DNA replication is quite similar. The two strands of cellular DNA are complementary in that the sequence of nucleotide bases in one determines the sequence of bases in the other. This follows from the Watson—Crick base-pairing rules. In the process of DNA replication, the following rules are useful to keep in mind:
2 The newly synthesized strand is antiparallel to its template.
3 New strands of DNA "grow" from the 5' to 3' direction.
The replication of a DNA molecule using the basic Watson—Crick rules is outlined in Fig. 13.1, where the proteins involved in prokaryotic as well as eukaryotic replication are noted. The parental DNA duplex "unwinds" at the growing point (the replication fork) and two daughter DNA molecules are formed. Each new daughter contains one parental strand and one new strand. Each base in the new strand and its polarity are determined by the three rules just presented.
The replication of DNA can be divided into two phases, initiation of a round of DNA replication, which leads to generation of a complete daughter strand, and the elongation of this
strand following initiation. Each round of DNA replication is initiated with a short piece of RNA that functions as a primer to begin the DNA chain. The first priming reaction takes place at a specific region (or site) on the DNA called the origin of replication (ori). This origin of replication comprises a specific sequence of bases where an origin-binding protein interacts and causes local denaturation to allow the replication process to begin.
Following denaturation of the DNA duplex at the origin of replication mediated by the ori binding protein, a primosome made up of helicase and primase enzymes synthesizes a short RNA primer in the 5' to 3' direction antiparallel to the DNA template, and a DNA melting enzyme (helicase) associates with the growing replication fork leading to unwinding of the template duplex. The process differs on the two template strands following the initiation of replication. One strand (the leading strand) is antiparallel to the growing DNA chain, and DNA polymerase and associated sliding clamp proteins remain associated with this template leading to the synthesis of a continuous strand of new DNA. The other strand, however, is in the wrong direction to serve as a template and remains single stranded by virtue of being stabilized by single stranded DNA binding proteins until a sufficient stretch of DNA forms. At this juncture, a primosome associates with the single-stranded DNA near the replication fork and a new primer is synthesized leading to association of DNA polymerase and associated clamp proteins and the synthesis of the second progeny strand of DNA (the lagging strand). As replication proceeds, enzymes and proteins required for unwinding the DNA duplex and maintaining it as single-stranded (ss) material interact with the denatured "bubble" to keep open the growing fork. Thus, at the DNA growing point, DNA synthesis is continuous in one direction, but discontinuous in the other direction. Lagging strand synthesis is required because in order to maintain proper polarity, new primer must be placed upstream of the growing point. In other words, priming must "jump" ahead on the template to continue synthesis of the new DNA strand, because DNA polymerase can only generate newly synthesized product in the 5' to 3' direction (reading the template strand 3' to 5'). As lagging strand synthesis proceeds, primer RNA must be removed, gaps repaired, and discontinuous fragments ligated together to make the full strand. These final steps require the action of an exonuclease for removing RNA primer and DNA ligase for linking together the fragments of growing DNA on the lagging strands.
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