DNA Replication

The metabolic processes described earlier (glycolysis, respiration, photophosphorylation, etc.), are dependent upon the enzymes present within cells. Most enzymes are proteins, (a few are RNA), and their presence within a cell is determined by the genetic information or hereditary material present. This material, contained primarily within the nucleus (eukaryotic cells) or nucleoid (prokaryotic cells), is deoxyribonucleic acid, commonly referred to as DNA.

Replication 

sometimes called semi-conservative replication, is the process involved when DNA molecules reproduce. It is a semi-conservative process in that each new DNA molecule formed contains half of the original molecule involved in the replication process (the original or "parental" strand). This is because during replication, each strand of the DNA duplex serves as a template or pattern for the new strand being formed. Given this feature, one might ask, just how old is DNA? 

Replication can occur by more than one mechanism, and is a complex process involving multiple factors not presented here. When considered in simplified form, replication always requires three things:

1.  An existing DNA molecule to serve as a pattern or template. 
2.  Enzymes – The heterogeneous proteins found in chromatin. 
3.  Energy – Because synthesis reactions are endergonic.

 Within living cells, DNA replication typically begins at a specific site called the origin of replication, and proceeds in both directions away from that point. Prokaryotic cells such as those of E. coli generally have only one origin of replication within their circular chromosome, but eukaryotic cells have many along their linear chromosomes. The origin of replication within an E. coli chromosome (called oriC), is a sequence of nucleotides 245bp in length. This region contains specific base sequences recognized by and able to interact with initiation factors and enzymes involved in the process. At the origin, the two, nucleotide strands of the DNA molecule separate (hydrogen bonds break) and individual bases are exposed between them. This separation involves enzymes e.g., helicases and gyrase (topoisomerase II). 

The primary enzyme involved in DNA replication is DNA-dependent DNA polymerase, often referred to simply as DNA polymerase. Prokaryotic cells such as E. coli typically have three DNA polymerase enzymes designated as DNA polymerase I, II and III. Of these, DNA polymerase III is the primary builder. Polymerase enzymes catalyze chemical reactions resulting in the formation of phosphodiester bonds, i.e., they synthesize polymers; however, DNA polymerase enzymes can only add nucleotides to the free, 3' ends of existing nucleotide chains (can build from 5' to 3'). They cannot initiate the formation of nucleotide strands from individual nucleotides without the presence of primers. A primer is a short sequence of nucleotides (often around 18-20 bases in length) and when associated with DNA replication is composed of RNA nucleotides (primers used in the PCR are often made of DNA). Enzymes called primase enzymes build the RNA primers associated with replication. The first of these is formed near the origin, and provides the free, 3' end DNA polymerase requires for synthesizing DNA. Once replication has been initiated, the DNA strands involved appear to form two replication forks, i.e., regions where the double helix separates into two, individual strands. These will travel in opposite directions (away from one another) as the original helix unwinds and replication proceeds. There is usually only one primer synthesized at the origin of replication, and it is associated with the leading strand. Once this primer is in place, DNA polymerase III can add DNA-type nucleotides to it and build a new complimentary strand (the leading strand) as a continuous sequence. The opposite strand forming the replication fork is called the lagging strand. Although nucleotides are also exposed along the lagging strand, replication cannot occur there in the same fashion because DNA polymerase cannot build in the 3' to 5' direction. Instead, a group of proteins including primase, form a structure called a primosome, and this begins to migrate along the lagging strand traveling in the same direction as DNA polymerase III (on the leading strand). 

Periodically, as the primosome reaches specific nucleotide sequences along the lagging strand, it synthesizes new primers (primer synthesis occurs in the 5' to 3' direction, so is opposite the direction of primosome migration). These primers (also made of RNA) serve as new start points for DNA synthesis, and initiate the formation of a series of DNA fragments called Okazaki fragments. Each Okazaki fragment has a short RNA sequence at its 5' end, but they are composed primarily of DNA. The Okazaki fragment formed nearest the origin serves as the beginning of the leading strand associated with the other replication fork (i.e., the one traveling in the opposite direction away from the origin). Since DNA molecules do not contain small segments of RNA, all the RNA primers formed during replication must be removed. This is accomplished by DNA polymerase I. It travels along the newly formed lagging strand, degrading the RNA primers and replacing them with DNA (it also removes the primer at the beginning of the leading strand). However, although DNA polymerase can add new nucleotides to a free, 3' end of an existing nucleotide strand, it cannot form a phosphodiester bond between two existing nucleotide strands. This requires a different enzyme called ligase. Ligase enzymes form the phosphodiester bonds attaching the multiple Okazaki fragments together, and bind the leading strand formed with one replication fork to the lagging strand formed with the other. Note – There is considerable "proof reading" and "repair" associated with the replication process, such that most newly formed DNA strands are identical to their "parental" compliments. Errors occur at a pace of about 1 per 100 million copies of DNA (the spontaneous mutation rate described in a later section). In addition to requiring DNA as a template, and the enzymes described above, replication also requires energy. Replication of the E. coli chromosome (around 4.6 X 106 bp), occurs in about 60 minutes, so proceeds at a pace of about 77 thousand nucleotides per minute (over 1000 per second). The process requires considerable energy, because the chemical reaction associated with the formation of each phosphodiester bond is endergonic. The energy required is provided by the nucleotides used in the building process, i.e., by dNTPs and rNTPs. Nucleoside triphosphates (NTPs) are high-energy molecules containing pyrophosphate bonds (recall the structure of ATP described in an earlier section). These bonds are broken as the nucleotides are incorporated into DNA, and the energy released is used to form the phosphodiester bonds holding the nucleotides together.