Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies
The extreme accuracy of the replication process is even more impressive when one considers the speed at which the cellular DNA can be replicated prior to cell division. Rapidly growing human cells can double about once every 24 hours. Ignoring the fact that only part of this time is used for DNA replication, this still translates to a rate of replication (for the entire genome) of nearly 50,000 nucleotides per second! Some other eukaryotes display even faster replication rates. For example, very early Drosophila fruit fly embryos replicate their genome in about 8 minutes, for an overall rate of over 600,000 nucleotides per second. These very high rates of replication can only be achieved by using multiple replication machines simultaneously, similar to having 5000 people each copying one of the imaginary Harry Potter books to complete the job in a shorter period of time. If you consider a single replication machine in the human system, the rate of replication is on the order of 30 base pairs per second, while bacterial replication machineries can achieve a rate of about 1000 base pairs per second. If human cell replication utilized only a single replication machine, it would take roughly 7 years to replicate all the DNA in one cell at the rate of 30 base pairs per second.
1.2The double-helical structure of DNA and the logic of replication and repair
In 1953, Watson and Crick published one of the most famous papers in biology, “A Structure for Deoxyribose Nucleic Acid” (see Further Reading at the end of this chapter). Critical research during the years leading up to this paper had provided evidence that DNA is the genetic material, and that the chemical composition of DNA consisted of purine and pyrimidine bases, a sugar called deoxyribose, and phosphates. A very important clue was that, for any particular DNA, the fractions of adenine and thymine bases were found to be identical, as were the fractions of guanine and cytosine bases (see “How did they test that” at the end of this chapter). Based on these prior studies and Rosalind Franklin’s X-ray crystallographic data, Watson and Crick deduced that the structure of DNA consisted of an elegant double helix with two linear strands wound around each other in opposite directions.
1962 Nobel Prize in Physiology or Medicine
This prize was awarded to Maurice Wilkins, James Watson, and Francis Crick for discovering the double helix structure of DNA and inferring the significance of this structure for information transfer and replication of the genetic material.
https://www.nobelprize.org/prizes/medicine/1962/summary/
Before delving into the implications of this structure for the logistics of replication, it is important to appreciate the DNA molecule. Individual strands of the double helix consist of a string of deoxyribonucleoside (or simply nucleoside in common parlance) monophosphates linked to each other in a linear fashion. As illustrated in Figure 1.1A–1.1C, a deoxyribonucleoside consists of a purine or pyrimidine base linked, via one of its nitrogen atoms, to the 1′-C atom of the deoxyribose sugar. This kind of linkage is called an N-glycosidic bond. The four bases in DNA are the purines adenine and guanine, and the pyrimidines thymine and cytosine. The corresponding deoxyribonucleosides, with sugar linked to the base, are named deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine (the RNA nucleosides are adenosine, uridine, guanosine, and cytidine). When a deoxyribonucleoside is also linked to one or more phosphate groups, it is called a deoxyribonucleotide (also referred to as deoxyribonucleoside monophosphate in the case of a single phosphate, deoxyribonucleoside diphosphate for two phosphates, etc.). The four common deoxyribonucleoside monophosphates that form the subunits of DNA are shown in Figure 1.1D (along with simple representations that will be used in figures throughout the book).
As mentioned above, the repeating unit in a DNA strand consists of deoxyribonucleoside monophosphate. Within each DNA strand, the monophosphate groups link together adjacent deoxyribose units by bonding to the 3′-C atom of one sugar and the 5′-C atom of its neighbor (corresponding to 3′-OH and 5′-OH groups in the unlinked deoxyribonucleoside; see Figure 1.1B). The linear nature of the strand results from the repeating sugar-phosphate units, called the sugar-phosphate backbone, while the bases are appended off of the sugar residues (Figure 1.2, right panel). The nature of the phosphodiester bond gives each DNA strand its directionality — the deoxyribonucleoside residue at each end of the DNA strand will have either a 3′-OH or a 5′-OH group that is not linked to a neighbor, and these residues define the 3′ end or the 5′ end.
Figure 1.1. Bases, nucleosides, and nucleotides. DNA is composed of repeating chains of linked nucleotides. The basic components of each nucleotide are a base (A), the sugar deoxyribose (B) (ribose in RNA), and phosphates (one phosphate per residue in DNA, three in the triphosphate precursor). The free base cytosine is shown in panel A, and the sugar deoxyribose in panel B, with the numbered carbon positions indicated. The combination of a base and sugar is a nucleoside, such as deoxycytidine (panel C, left), with the base connected to the 1′-carbon of the sugar. The addition of one or more phosphates to the 5′-carbon of the nucleoside creates a nucleotide, such as deoxycytidine monophosphate (panel C, right). The four major nucleoside monophosphates in DNA are shown in panel D, along with a diagrammatic representation of each that will be used in various figures throughout this book. (Hydrogens on the carbon atoms in the bases and on the 5′-C of the sugars are not shown.)
In the double helix of DNA, the two involved strands are in opposite orientation, or “anti-parallel,” with one sugar-phosphate backbone oriented in the 5′ to 3′ direction and the other 3′ to 5′ (Figure 1.2). The inherent beauty of the DNA double helix is that the four bases partner with each other from opposite strands — adenine (A) always pairs with thymine (T) and guanine (G) always pairs with cytosine (C) — the four canonical base pairs in duplex DNA (A:T, T:A, G:C, and C:G).2 This pairing is made possible in the context of the two strands winding, in a right-handed helical fashion, around each other about once every 10.5 base pairs (Figure 1.2, left panel, middle, and bottom). The paired bases are on the interior of the duplex, while the sugar-phosphate backbone dominates the exterior.
As shown in Figure 1.3, the pairing of the bases involves a particular arrangement of hydrogen bonds across the helix (three bonds for G:C or C:G and two for A:T or T:A). The interior bases of the double helix are “stacked” on top of each other, providing stability. The overall shape of the helix provides two grooves, called the major and minor groove, which expose the edges of the bases and allow proteins to recognize particular sequences without needing to unwind the two strands of the double helix (Figure 1.2, bottom).
With this appreciation of the structure of DNA, we can now consider how this structure impacts the processes of DNA replication and repair. In their 1953 paper, Watson and Crick proposed how the double-helical structure resolves the puzzle of the duplication of the genetic material, with their famous line: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” They realized that the two strands of DNA contain, in a sense, redundant information — either strand could direct an entirely new double helix, identical to the parental duplex, by using the rules of base pairing to recreate the opposite strand. If both single strands of the parental are used in this way as templates for their new partner strand, the result is two daughter duplexes that have the same sequence as each other and as the parental duplex. Note that each daughter duplex consists of one strand of parental DNA and one strand of new DNA, hence the process is characterized as being “semi-conservative” (one strand conserved, one new).