4.1). This RNA chain, the primer, is needed to prime (or start) the synthesis of the new DNA strand. DNA polymerase III catalyzes the formation of a phosphodiester bond between the 3′‐hydroxyl group of the RNA primer and the 5′‐phosphate group of the appropriate deoxyribonucleotide. Several RNA primers are made along the length of the lagging strand template. Each is extended in the 5′ to 3′ direction by DNA polymerase III until it reaches the 5′ end of the next RNA primer. In prokaryotes the lagging strand is primed about every 1000 nucleotides whereas in eukaryotes this takes place every 200 nucleotides.
RNA Primers Are Removed
Once the synthesis of the DNA fragment is complete, the RNA primers must be replaced by deoxyribonucleotides. In prokaryotes the enzyme DNA polymerase I removes ribonucleotides using its 5′ to 3′ exonuclease activity and then uses its 5′ to 3′ polymerizing activity to incorporate deoxyribonucleotides. In this way, the entire RNA primer is replaced by DNA. Synthesis of the lagging strand is completed by the enzyme DNA ligase, which joins the DNA fragments together by catalyzing the formation of phosphodiester bonds between adjacent fragments.
Eukaryotic organisms use an enzyme called ribonuclease H to remove their RNA primers. These enzymes break phosphodiester bonds in an RNA strand that is hydrogen‐bonded to a DNA strand. Within each cell, ribonuclease H2 is involved in removing RNA primers during replication of nuclear genomic DNA and ribonuclease H1 is involved in removing RNA primers during replication of mitochondrial genomic DNA.
The Self‐Correcting DNA Polymerase
The genome of E. coli consists of about 4.6 × 106 base pairs of DNA. DNA polymerase III makes a mistake about every 1 in 105 bases and joins an incorrect deoxyribonucleotide to the growing chain. If unchecked, these mistakes would lead to a catastrophic mutation rate. Fortunately, DNA polymerase III has a built‐in proofreading mechanism that corrects its own errors. If an incorrect base is inserted into the newly synthesized daughter strand, the enzyme recognizes the change in shape of the double‐stranded molecule, which arises through incorrect base pairing, and DNA synthesis stops (Figure 4.2b). DNA polymerase III then uses its 3′ to 5′ exonuclease activity to remove the incorrect deoxyribonucleotide and replace it with the correct one. DNA synthesis then continues. DNA polymerase III hence functions as a self‐correcting enzyme.
Mismatch Repair Backs Up the Proofreading Mechanism
The proofreading function of DNA polymerase III improves the accuracy of DNA replication about 100‐fold. However, sometimes the enzyme does miss a nucleotide that has been incorrectly inserted into the newly synthesized DNA strand. Cells have evolved a backup mechanism, mismatch repair, that detects when an incorrect nucleotide has been inserted into the daughter strand (Figure 4.3). The repair mechanism relies on the cell being able to distinguish, within the double helix, between the template strand (the parental strand) and the newly synthesized strand (the daughter strand).
We best understand this repair process in E. coli . The bacterium has an enzyme called Dam methylase that adds a ‐CH3 group, called a methyl group, onto the A of the sequence 5′ GATC3′. This sequence occurs very frequently in DNA, about once every 256 bp. The methylation of DNA happens very soon after a DNA strand has been replicated. However, for a short time during replication the double‐stranded DNA molecule will have one strand methylated (the parental strand) and one strand not methylated (the daughter strand). The DNA molecule is said to be hemi‐methylated (half methylated). Because the newly synthesized strand has not yet been methylated the cell knows that if a mismatch in base pairing has occurred between the two strands it is the nonmethylated, newly synthesized, strand that must carry the mistake.
A protein called MutH binds on the newly synthesized strand at a site opposite a methylated A in the template strand. If there is no mismatched base pair nearby then MutH does nothing. However, if two other proteins called MutL and MutS have detected a mismatched base pair then MutH, which is an endonuclease, is activated and nicks (cleaves a phosphodiester bond between two nucleotides in) the unmethylated newly synthesized strand. This allows a stretch of DNA containing the mismatched base pair to be removed. Two different proteins are involved in removing the stretch of DNA. If MutH nicks the DNA 5′ to the mismatch (Figure 4.3a), then exonuclease VII degrades the DNA strand in the 5′ to 3′ direction. However, if MutH nicks the DNA 3′ to the mismatch (Figure 4.3b), then the DNA strand is removed by exonuclease I in the 3′ to 5′ direction. In either case, the gap in the daughter strand is then replaced by DNA polymerase III.
Deoxyribonucleic acid can be damaged by a number of agents, which include oxygen, water, naturally occurring chemicals in our diet, and radiation. Because damage to DNA can change the sequence of bases, a cell must be able to repair alterations in the DNA code if it is to survive and pass on the DNA database unaltered to its daughter cells.
Spontaneous and Chemically Induced Base Changes
The most common damage suffered by a DNA molecule is depurination – the loss of an adenine or guanine because the bond between the purine base and the deoxyribose sugar to which it is attached spontaneously hydrolyzes (Figure 4.4). Within each human cell about 5000–10 000 depurinations occur every day.
Deamination is a less frequent event; it happens about 100 times a day in every human cell. Collision of H3O+ ions with the bond linking the amino group to carbon number 4 in cytosine sets off a spontaneous deamination that produces uracil (Figure 4.4). Cytosine base pairs with guanine, whereas uracil pairs with adenine. If this change were not corrected, then a CG base pair would mutate to a UA base pair the next time the DNA strand was replicated, introducing a mutation at this position in one of the two copies of the DNA double helix that are obtained post‐replication.
Ultraviolet light or chemical carcinogens such as benzopyrene, present in cigarette smoke, can also disrupt the structure of DNA. The absorption of ultraviolet light can cause two adjacent thymine residues to link and form a thymine dimer (Figure 4.5). If uncorrected, thymine dimers create a distortion in the DNA helix known as a bulky lesion. This inhibits normal base pairing between the two strands of the double helix and blocks the replication process. Ultraviolet light has a powerful germicidal action and is widely used to sterilize equipment. One of the reasons why bacteria are killed by this treatment is because the formation of large numbers of thymine dimers prevents replication.