Nucleic Acids and Molecular Genetics

Huntington’s Disease, A Single-Gene Mutation

There are 4000 or more human diseases caused by a single-gene mutation. Huntington’s disease is an example of an autosomal dominant disease . There are many others like this: Marfan syndrome, neurofibromatosis, retinoblastoma, myotonic dystrophy, familial hypercholesterolemia, adult polycystic kidney disease, familial adenomatous polyposis, hypertrophic obstructive cardiomyopathy , and osteogenesis imperfecta to name a few. In autosomal recessive diseases , one in four offspring will be overt bearers of the disease, two of four children will be silent carriers, and one child will be perfectly normal, whereas in autosomal dominant diseases , each child has a 50% chance of inheriting the disease with no skipping of generations. Only one parent needs to have the dominant allele. Only rarely is the (sporadic) disease manifest in a person whose parents do not have the mutation. The pedigree in two successive generations from a husband and wife, where the male has the dominant gene, is shown in Fig. 10.1 .

Huntington’s is a rare disease affecting 1 person of European descent in 10,000. Death occurs in one person in 600,000. The disease is less common among Chinese, Japanese, and African descendants. There are childhood and adult onset forms of the disease. The early onset of the disease progresses more rapidly than the adult onset. Early onset occurs in childhood or adolescence, and these individuals live 10 or 15 years after disease symptoms (movement problems, mental and emotional changes, clumsiness, falling down, slurred speech, and drooling) occur. Up to 50% of diseased children have seizures . Suicides can occur when the disease strikes in the second decile. The most common form of Huntington’s is the adult onset form, which has many of the symptoms of the early onset. Involuntary jerking or twitching in the adult form is referred to as Huntington’s chorea (from the Greek “khoreia” meaning choral dance). Adults live for 15–20 years after symptoms begin.

The human gene involved is the HTT gene that is transcribed to messenger ribonucleic acid (mRNA) that translates to a protein called Huntingtin , which, in mice, has been shown to have a role in the normal functioning of the basal ganglia . Huntingtin is expressed in all types of brain neurons and is a soluble protein of about 350 kD having 3144 amino acids and containing about 36 alpha-helical HEAT (His–Glu–Ala–Thr) repeats . Although the exact function of this protein is unknown, experiments in which the equivalent gene to human Htt in mice (Hdh) is deleted, there is a suppression of the calcium ion mobilization response to inositol trisphosphate . This suggests that Huntingtin plays a direct role in neuronal Ca ²⁺ signaling by reducing the sensitivity of the inositol trisphosphate receptor in the endoplasmic reticulum to its ligand, inositol tris phosphate. Deletion of this gene in mice is embryonic lethal.

The HTT gene contains a chicken β-actin/globin (CAG) trinucleotide repeat segment. This trinucleotide is repeated 10–35 times within the gene, coding for polyglutamine . In Huntington’s disease the CAG segment is repeated 36 to an excess of 120 times. While individuals with 27–35 CAG repeats in the HTT gene do not develop the disease, their offspring may develop the disease. An intermediate form exists in which the CAG segment is repeated 36–40 times and these individuals may or may not develop the symptoms. Those who have an excess of 40 repeats in the HTT gene always develop the disease. When the number of CAG repeats exceeds 60, juvenile onset is predicted, whereas mutations showing 40–55 repeats result in adult onset. The CAG triplet repeat is defined as a short tandem repeat polymorphism . In experimental animals the normal huntingtin (Htt) plays a role in the development of the basal ganglia in the brain. In the abnormally elongated version of huntingtin (Htt*) in neurons, the pathologic proteins form and are broken down enzymatically into smaller toxic fragments that aggregate and disrupt cellular functioning and eventually cause the death of the cell ( Fig. 10.2 ).

Another analysis indicates that there is reduced brain-derived neurotrophic factor ( BDNF ) in Huntington’s disease. Htt* disrupts BDNF induction of the mitogen-activated protein kinase ( MAPK ) by reduction in the expression of adapter proteins ( p52/46Shc ) and the interference with Ras activation . This leads to a reduction in the growth pathway through ERK1/2 , or MAPK, and tips the balance between cell survival and cell death in favor of cell death. These relationships are shown in Fig. 10.3 .

Other evidence involves a direct effect of Htt in the pretranscriptional complex . The transcriptional factor Sp1 (specificity protein 1) binds to DNA elements (GC boxes) in cellular promoters. A protein–protein interaction between the glutamines of Htt and the diglutamines of Sp1 and TAF II 130 subunit (Tata-associated factor 130, cofactor for NFAT) is needed to recruit the transcriptional machinery (other proteins, including RNA polymerase II ). In Huntington’s disease, with the extension of polyglutamine in Htt*, RNA polymerase is not able to bind well and transcription does not take place. This work has been done in the context of the transcription of the dopamine receptor . (Reproduced from http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/McDonald/Huntington.htm .) A transgene (a gene placed experimentally in the germline that functions as a normal gene) in mice containing expanded polyglutamine tracts generates the central nervous system disorder resembling Huntington’s disease, whereas a transgene with a normal number of polyglutamines does not cause the disease. In addition to Huntington’s disease, spinobulbar muscular atrophy shows expansion of the CAG triplet expansion in the gene for the androgen receptor . The fragile X syndrome is marked by an expansion in a polyarginine tract ( CGG triplet expansion) and there are other diseases in this category.

An abnormal protein followed by an aggregation phenomenon could classify Huntington’s disease as a familial prion disease . The alteration of the gene ( HTT ) to the disease form ( HTT* ) occurs during the development of the sperm . Neuronal loss in the neostriatum and the cortex of the brain occurs at age 35–44 years, the variation depending on the state of methylation of the HTT locus. Neuronal loss leads to a decrease in metabolic activity of the brain ( Fig. 10.4 ), especially where the uptake and metabolism of glucose are concerned.

Detection of the disease can be made with a blood sample by sequencing the CAG repeat region in the DNA.

Purines and Pyrimidines

Purines and pyrimidines make up the bases in both RNA and DNA. The two common purines are adenine and guanine . Purine structure consists of two adjoined rings with five carbons and four nitrogens. The common pyrimidines are cytosine , thymine , and uracil . Pyrimidines have one ring with four carbons and two nitrogens. The structures are shown in Fig. 10.5 .

In the nucleotide form in DNA, adenine, guanine, cytosine, and thymine attach to deoxyribose , whereas in RNA, adenine, guanine, cytosine and uracil attach to ribose. In RNA, uracil replaces thymine because if uracil appeared in DNA, it would be recognized by the base excision repair mechanism and it would be removed. The combination of a purine or a pyrimidine with a sugar is a nucleoside . When the sugar is phosphorylated, it is a nucleotide . One phosphate group renders it a mononucleotide (this form is contained in RNA and DNA); two phosphate groups, a dinucleotide and three phosphate groups, a trinucleotide. In nucleic acids, these bases are bound with ribose (RNA) or deoxyribose (DNA) and they are phosphorylated (mononucleotides). The nucleosides (nonphosphorylated) appear as shown in Fig. 10.6 . Nucleotides are more prevalent in the cell than nucleosides.

Deamination of cytosine to form uracil can occur through accident or by a mutation as shown in Fig. 10.7 .

If this occurs in DNA, uracil will be base-paired with guanine and uracil could be removed directly and replaced by cytosine for the correct C–G base pairing. In rare cases, uracil could be paired with adenine after replication, and a repair mechanism could replace the uracil with a thymine to give a T–A base pair resulting in a mutated DNA. Base pairing will be discussed. Deamination of other bases can occur: guanine can be deaminated to form xanthine, and adenine to form hypoxanthine. Xanthine and hypoxanthine are metabolic products and do not generally appear in nucleic acids. Orotic acid , a pyrimidine carboxylic acid, is a metabolite. In nucleosides and nucleotides, the base can exist in two possible orientations about the N -glycosidic bond ( Fig. 10.8 ). The anti form is the one that predominates in nucleic acids.

The structures and naming of the bases and their derivatives appear in Fig. 10.9 , where part (A) shows the naming of the base alone, the monosaccharide derivative, and the monosaccharide monophosphate derivative. Fig. 10.9B shows the potential structures of the derivatives of adenosine or deoxyadenosine. Fig. 10.9C shows the structures of a common pyrimidine (orotic acid) and of hypoxanthine and xanthine (purines), all of which are metabolites rather than members of RNA or DNA. Orotic acid is an important intermediate in pyrimidine synthesis. It has been labeled as vitamin B13 because it can partially compensate for vitamin B12 deficiency and is active in folic acid metabolism.

If a nucleoside or nucleotide contains deoxyribose as compared to ribose, its designation is preceded by a d , as in dAMP.

There are cyclic forms of adenosine and guanosine: these are cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (GMP). cAMP is formed from adenosine triphosphate (ATP) by the action of adenylate cyclase and cyclic GMP is formed from guanosine triphosphate (GTP) by the action of guanylate cyclase ( Fig. 10.10 ). These are separate enzymes. Adenylate cyclase is a 12-transmembrane protein, while guanylate cyclase is a single-transmembrane protein. Guanylate cyclase is part of a receptor for the natriuretic peptide , a hormone (Chapter 11: Protein Biosynthesis ), and there are four related receptors. The enzymatic activity of a cyclase is classified as a lyase (Chapter 5: Enzymes ).

Base Pairing

Adenine forms a base pair with thymine in DNA and with uracil in RNA. Guanine pairs with cytosine. Base pairing is shown in abbreviated fashion in Fig. 10.11 .

The pairs of bases are held together by hydrogen bonding . Other forces contribute to the stability of the double-stranded DNA. There are ionic charges (van der Waals forces) between the tightly stacked pairs and hydrophobic attractions occur between base pairs in the interior of the helix as the nonpolar nitrogenous bases are packed so tightly to exclude water molecules and form a stable nonpolar interior. The bases are located in the interior of the double-stranded DNA and the pentose sugars, and phosphate groups are on the outside as shown in the chemical structures in Fig. 10.12 . van der Waals forces usually involve dipolar molecules where the concentration of positive charges is separated from a concentration of negative charges so that there are exerted attractive (or repulsive) forces between molecules (that do not arise from covalent or ionic bonds).

The 3′ carbon of 1 sugar is linked to the 5′ carbon of the next sugar through a phosphodiester bond. A single turn of the DNA helix (of the naturally occurring abundant B-DNA) requires 10 base pairs and the height of the turn measures 3.4 nanometers (nm); the diameter of the helix measures 1.9 nm. The double helix is formed by two antiparallel DNA strands. The 5′-end of the left strand is at the top of the figure and the 3′-end is on the bottom. Replication of DNA and transcription to form mRNA from the sense strand of DNA both proceed in the direction from the 5 ′- end to the 3 ′- end .

The Structure of DNA

A small portion of a single strand of DNA (AGACC) is shown in Fig. 10.13 . Deoxyribose, a nucleoside component and the phosphate backbone, is indicated.

Transcription of DNA to form the corresponding mRNA proceeds from the 5′-end to the 3′-end of the RNA molecules. Normally, DNA resides in the cellular nucleus with two strands in the form of a helix. One strand of DNA would appear as shown in Fig. 10.13 and the second strand would be antiparallel where the bases in both strands are joined together by base pairing, each pair of bases being held together by hydrogen bonding. Double-stranded DNA appears as in Fig. 10.14 .

Double-stranded DNAs of any length appear as helices, that is, the two strands together are twisted into a helix as shown in Fig. 10.15 .

Again, the base pairing in DNA is dR-A-T-dR (or R-A-U-R in RNA), and dR-G-C-dR (or R-G-C-R in RNA). dR is deoxyribose and R is ribose. Although not connoted here, phosphate groups are attached to the sugar residues. DNA double strands can be melted apart into single strands in vitro by heating to 50°C–60°C. Hydrogen bonds as well as the other forces holding the two strands together are loosened as the temperature is increased. The individual chains reform the double helix when the temperature is returned to normal at about 37°C. The melting temperature is an important characteristic of each double-stranded DNA molecule. The preponderant form of DNA in the cell is the B form that was originally described by Watson and Crick ( Fig. 10.15B ). Three forms are known in addition to the B form. Forms A, B, and C each has a right-handed helix (chains proceed from the bottom toward the top in a series of right-hand turns), while Z-DNA has a left-handed helix (from the bottom to the top the chains proceed in a series of left-hand turns). The Z form is found in vitro for the most part under nonphysiological high salt conditions; however, Z forms can occur in the cell along with other strands of the B form when there are stretches of poly GC or poly AT or nearby methylcytosine. These conditions apparently can stabilize runs of Z-DNA. For comparison the structures of A-DNA, B-DNA , and Z-DNA are shown in Fig. 10.16 .

The haploid human genome consists of 24 pairs of chromosomes (22 autosomal chromosomes plus XX sex chromosomes in the female and XY sex chromosomes in the male). All cells of the body contain the complete genome, except for the red blood cell that does not have a nucleus and, therefore, contains no chromosomes. The microscopic spread of chromosomes is shown in Fig. 2.14. Each chromosome varies in its number of base pairs from about 50 to 250 million. The human genome consists of about 3 billion base pairs. Genes account for about 2% of the human genome and encode the information for about 30,000 genes. Usually there are long stretches of noncoding DNA that separate the genes. Aside from that DNA, much of the DNA outside of the genes has been ascribed a few specific functions.

Biosynthesis of Purines and Pyrimidines

Purines are derived from aspartate , carbon dioxide , glycine , N ¹⁰ -formyl-tetrahydrofolate ( N ¹⁰ -formyl-THF), and glutamine as diagramed in Fig. 10.17 .

The biosynthetic route begins with ribose-5-phosphate that is converted to 5-phospho- d -ribosyl-1-pyrophosphate ( PRPP ) in a reaction catalyzed by PRPP synthase ( Fig. 10.18 ). PRPP is the starting substrate for purine biosynthesis .

In all, there are 11 enzymatic steps leading to the formation of inosine-5 ′ -monophosphate ( IMP ). The 11 catalytic steps are described briefly in Fig. 10.19 where the structures of only the initial substrate and final product are given and the name of the enzyme involved in each step is given in the figure legend.

The synthesis of the purine nucleotide, IMP , makes possible the conversion to two major purine nucleotides, AMP and GMP , as shown in Fig. 10.20 .

Adenylosuccinate is formed from IMP as an intermediate by the addition of aspartate and donation of a phosphate group from GTP and then fumarate is split out to form AMP. GMP is formed from IMP with the addition of water and participation of oxidized nicotinamide adenine dinucleotide to form xanthosine monophosphate . Then glutamine, water, and ATP participate to form nucleotides [ADP and guanosine diphosphate (GDP)] and trinucleotides (ATP and GTP). In Fig. 10.21 the feedback and feed-forward effects of the various components of the system are seen.

The pyrimidine structure is derived from the amide group of glutamine, aspartate, and HCO ₃ ⁻ . Carbamoyl phosphate synthase II (CPSII) catalyzes the synthesis of carbamoyl phosphate from 2ATP, HCO ₃ ⁻ , glutamine, and water. In a series of reactions involving the enzymes: CPSII, aspartate transcarbamoylase ( ATCase ), dihydroorotase , dihydroorotate dehydrogenase , orotate phosphoribosyltransferase , and orotidine monophosphate (OMP) decarboxylase , the pyrimidine mononucleotide, uridine monophosphate (UMP) is derived ( Fig. 10.22 ).

CPSII is present in most tissues outside of the liver. There is at least one other enzyme, carbamoyl phosphate synthase I , that is involved in the urea cycle. It catalyzes the formation of carbamoyl phosphate from ammonia and bicarbonate in the mitochondrion.

Once UMP is formed, it can be converted to UDP by nucleoside monophosphate kinase :

UDP can be phosphorylated further to form the trinucleotide:

UTP can be converted to the cytidine triphosphate by citrate transport protein (CTP) synthase as shown in Fig. 10.23 .

The amine group of cytosine is derived from the amide group of glutamine . The purine trinucleotide GTP activates CTP synthase so as to affect a balance between pyrimidine and purine nucleotides in the cell. CTP synthase is reversibly inhibited by the product of the reaction, CTP, which also inhibits by product feedback , the formation of carbamoyl phosphate as shown in the overall pyrimidine biosynthetic pathway ( Fig. 10.24 ).

Orotic aciduria is a disorder associated with pyrimidine metabolism. There are variations of this disorder but the cause is a defective OMP decarboxylase , normal function of which would yield UMP from OMP ( Fig. 10.24 ). This condition can result in growth retardation, megaloblastic anemia, and leukopenia. Drugs, such as allopurinol (used for gout to block xanthine oxidase activity) and 6-azauridine , produce products that inhibit OMP decarboxylase.

Purine Interconversions

The formation of PRPP from ribose-5-phosphate initiates the purine synthetic pathway to generate phosphoribosylpyrophosphate (PRPP) . Nine enzymatic steps follow to finally produce inosine-5′-monophosphate ( IMP ) ( Fig. 10.19 ). IMP, the primary product of purine biosynthesis, can be converted to adenine and guanine nucleoside monophosphates ( Fig. 10.25 ).

IMP is first converted to adenylosuccinate catalyzed by adenylosuccinate synthase . This is converted to AMP by adenylosuccinate lyase . For conversion to GMP , IMP is converted to xanthosine monophosphate by the action of IMP dehydrogenase . Xanthosine monophosphate is converted to GMP by GMP synthase . These mononucleotides can be converted to the dinucleotides by the appropriate kinase:

by guanylate kinase, and

by adenylate kinase , and nucleoside diphosphates can be converted to the triphosphates by nucleoside diphosphate kinase , for example (also Fig. 10.21 )

Catabolism of Purine and Pyrimidine Nucleotides

The purine derivatives AMP and IMP are degraded by nucleotidase that removes the phosphate group to yield the corresponding nucleoside. The sugar is then removed by the action of nucleotide phosphorylase to generate the free bases, adenine or hypoxanthine . Adenine can be deaminated to form hypoxanthine as well. The resulting hypoxanthine is oxidized by xanthine oxidase to form xanthine that is further oxidized by xanthine oxidase to form uric acid , the ultimate product of purine nucleotide catabolism. Likewise, guanine can also be deaminated and the product subsequently oxidized to form uric acid. Thus xanthine oxidase is the terminal enzyme in the catabolic pathway, and uric acid is the final circulating product. As has been mentioned, too high a level of circulating uric acid can result in its crystallization in toe and other joints producing the disease of gout . The catabolic pathway for purine nucleotides is shown in Fig. 10.26 .

Salvage Pathway

Catabolism of purines generates purine bases and hypoxanthine as the penultimate product. Adenine, guanine, and hypoxanthine can be recovered through a salvage pathway by phosphoribosylation . The enzymes involved in the recovery process are adenine phosphoribosyltransferase ( APRT ) and hypoxanthine-guanine phosphoribosyltransferase ( HGPRT ). The reaction catalyzed by APRT is as follows:

The reactions catalyzed by HGPRT are

and

The conversion of IMP to AMP and the salvage of IMP from the catabolism of AMP involve the deamination of aspartate to fumarate through the purine nucleotide cycle shown in Fig. 10.27 .

For thymine nucleotides (thymine is exclusive to DNA, while uracil is exclusive to RNA), the sugar is deoxyribose and it appears in DNA as deoxythymidylate monophosphate (dTMP) . dTMP can be formed from dUMP by thymidine kinase that can catalyze the following reactions:

or

dUMP can be converted to dTMP by the action of thymidylate synthase as shown in Fig. 10.28 .

Pyrimidine Catabolism

A product of pyrimidine catabolism is β-alanine, derived from uracil. Uracil is a product of the breakdown of CMP and UMP (CMP can be converted to UMP). CMP and UMP derive from RNA catabolism . β-Aminoisobutyrate derives from the breakdown of DNA through dCMP and dTMP to evolve thymine. These reactions are shown in Fig. 10.29 .

During the catabolism of RNA, uracil is released ( Fig. 10.29 ) and it can be salvaged back to UMP by uridine phosphorylase and uridine kinase :

$\mathrm{uracil}+\text{ribose}-1-\text{phosphate}\stackrel{\mathrm{uridine}\mathrm{phosphorylase}}{\leftrightharpoons }\mathrm{uridine}+\mathrm{Pi}$

and

$\mathrm{uridine}+\mathrm{ATP}\stackrel{\mathrm{uridine}\mathrm{kinase}}{\leftrightharpoons }\mathrm{UMP}+\mathrm{ADP}$

Thymine can also be salvaged in similar fashion:

$\mathrm{thymine}+\mathrm{deoxyribose}-1-\mathrm{phosphate}\stackrel{\mathrm{thymine}\mathrm{phosphorylase}}{\leftrightharpoons }\mathrm{thymidine}+\mathrm{Pi}$

and

$\mathrm{thymidine}+\mathrm{ATP}\stackrel{\mathrm{thymidine}\mathrm{kinase}}{\leftrightharpoons }\mathrm{dTMP}+\mathrm{ADP}$

Thymidine kinase is an important enzyme; its concentration changes during the cell cycle and is at its peak during DNA synthesis.

Deoxycytidine kinase allows for the salvage of deoxycytidine , its preferred substrate. However, deoxyadenosine and deoxyguanosine are also substrates for this enzyme. The reaction with deoxycytidine is as follows:

$\mathrm{deoxycytidine}+\mathrm{ATP}\stackrel{\mathrm{deoxycytidine}\mathrm{kinase}}{\rightleftharpoons }\mathrm{dCMP}+\mathrm{ADP}$

Because of the low concentrations of both ribose-1-phosphate and the pyrimidine nucleosides, this pathway is a minor one for the salvage of pyrimidines.

dTMP can be formed from dUMP as shown in Fig. 10.28 . The reaction is catalyzed by thymidylate synthase . 5,10-Methylene THF coenzyme donates a methyl group to dUMP to form dTMP.

Deoxyribose-Containing Nucleotides

The syntheses of deoxyribose trinucleotides needed for the synthesis of DNA are summarized in Fig. 10.30 .

Disorders of Purine and Pyrimidine Metabolism

The end product of purine metabolism is uric acid . Frequently, the level of uric acid in plasma is high and this condition can lead to gout (normal uric acid concentration , 3.6–8.3 mg/dL; levels as high as 9.6 mg/dL can occur without the generation of gout). Levels of plasma uric acid can be high enough ( hyperuricemia ) to cause crystallization in various joints that is common in the ball joint of the large toe. Crystals can form in the kidney (sometimes leading to kidney stones ) and in capillaries. High blood creatinine is associated with high uric acid and may reflect decreased glomerular filtration . Gout is considered to be a form of arthritis and some believe that high plasma uric acid is a predictor of cardiovascular disease. Curiously, uric acid is an antioxidant, the highest level of an antioxidant in blood. It may be an indicator of oxidative stress. Uric acid is quite insoluble in water, whereas its metabolite, (S)-allantoin , is 10 times more water-soluble than uric acid. It is unclear whether uric acid is actually functioning as an antioxidant in blood. Uric acid metabolism occurs in the peroxisome by urate oxidase (or by catalase) and through two intermediates, produced through two other enzymes, leads to (S)-allantoin . There can be genetic alterations in the genes for these enzymes that can account for high circulating uric acid and such studies are underway. High uric acid can be treated with the drug allopurinol that is a competitive inhibitor of xanthine oxidase ( Fig. 10.31 ); however, in some cases of gout, the enzyme urate oxidase has been used effectively as a treatment which seems superior to allopurinol unless an allergic reaction to the enzyme protein develops.

Diseases associated with disorders of purine or pyrimidine metabolism are listed in Table 10.1 .

Table 10.1

Diseases Associated With Disorders of Purine or Pyrimidine Metabolism.

Disorder	Defect	Nature of Defect	Comments
Gout	PRPP synthetase	Increased enzyme activity due to elevated V max	Hyperuricemia
Gout	PRPP synthetase	Enzyme is resistant to feedback inhibition	Hyperuricemia
Gout	PRPP synthetase	Enzyme has increased affinity for ribose-5-phosphate (lowered K m )	Hyperuricemia
Gout	PRPP amidotransferase	Loss of feedback inhibition of enzyme	Hyperuricemia
Gout	HGPRT	Partially defective enzyme	Hyperuricemia
Lesch–Nyhan syndrome	HGPRT	Lack of enzyme
SCID	ADA	Lack of enzyme
Immunodeficiency	PNP	Lack of enzyme
Renal lithiasis	APRT	Lack of enzyme	2,8-Dihydroxyadenine renal lithiasis
Xanthinuria	Xanthine oxidase	Lack of enzyme	Hypouricemia and xanthine renal lithiasis
von Gierke’s disease	Glucose-6-phosphatase	Enzyme deficiency
Pyrimidine Metabolism
Disorder	Defective Enzyme	Comments
Orotic aciduria, type I	Orotate phosphoribosyltransferase and OMP decarboxylase
Orotic aciduria, type II	OMP decarboxylase
Orotic aciduria (mild, no hematological component)	The urea cycle enzyme, ornithine transcarbamoylase, is deficient	Increased mitochondrial carbamoyl phosphate exits and augments pyrimidine biosynthesis; hepatic encephalopathy
β-Aminoisobutyric aciduria	Transaminase, affects urea cycle function during deamination of α-amino acids to α-keto acids	Benign, frequent in Asians
Drug-induced orotic aciduria	OMP decarboxylase	Allopurinol and 6-azauridine treatments cause orotic acidurias without a hematological component; their catabolic by-products inhibit OMP decarboxylase

ADI , Adenosine deaminase; APRT , adenine phosphoribosyltransferase; HGPRT , hypoxanthine-guanine phosphoribosyltransferase; OMP , orotidine monophosphate; PNP , purine nucleotide phosphorylase; PPRP , 5-phospho- d -ribosyl- l -pyrophosphate; SCID , severe combined immunodeficiency.

Hypoxanthine-guanine phosphoribosyltransferase ( HGPRT ) is an important enzyme in the purine salvage pathway . It catalyzes the conversion of hypoxanthine to inosine monophosphate ( IMP ) and the conversion of guanine to GMP . Thus it plays a major role in generating purine nucleotides through the purine salvage pathway (see the “Salvage Pathway” section). It is possible to lose the function of this enzyme, located on the X chromosome. This trait translates into patients, primarily males, with a relatively rare recessive disease characterized by severe gout and a central nervous system disorder ( Lesch–Nyhan syndrome ; see Table 10.1 ). The disease is independent of geography and race and occurs in one of 380,000 births. The severe form of this disease is characterized by self-mutilation . There is no direct treatment for this condition except to use devices that will limit self-mutilation and therapy for gout, primarily the use of allopurinol.

Diseases associated with pyrimidine metabolic disorders are not as problematical as those associated with the dysfunction of purine metabolism because the products are more water-soluble than uric acid. Some of these diseases are described in Table 10.1 .

Biosynthesis of Deoxyribonucleic Acid in the Nucleus

The structure of DNA and the nature of the individual strands of DNA have been discussed ( Figs. 10.12–10.16 ). DNA is a large polynucleotide. In the haploid number (23) of human chromosomes, their length is about 3 billion base pairs with a content of about 25,000 different genes. The earliest form of genetic information may have been in the form of RNA that could have developed before the existence of DNA.

Some evidence suggests that the beginning of DNA synthesis occurs in a small number of perinucleolar sites (within the nucleus) that are regulated coordinately and which are selected in the G1 phase of the cell cycle. In forming the DNA strand, the phosphate group of a mononucleotide reacts with the free 3′-hydroxyl group of a second nucleotide to form a dinucleotide joined through a phosphoric acid ester , and the DNA strand is built by a continuation of this process ( Fig. 10.32 ).

Given that there is an existing duplex of template and primer, DNA polymerase acts. The first nucleotide in the strand will have a free 5′-phosphate group and the last nucleotide added in the completed strand will have a free 3′-hydroxyl ( Fig. 10.13 ); thus information in the DNA strand will proceed from the 5′-end to the 3′-end. A hypothetical strand of DNA could be written (starting from the 5′-end):

$\begin{array}{ccc}5\prime & \mathrm{ATGCTACGC}& 3\prime \end{array}$

and the antiparallel strand together with the previous strand would generate the double strand:

$\begin{array}{ccc}5\prime & \mathrm{ATGCTACGC}& 3\prime \\ 3\prime & \mathrm{TACGATGCG}& 5\prime \end{array}$

remembering that each letter represents a deoxyribose mononucleotide.

In the case of an mRNA formed from the previous DNA sequence which is

$5\prime \mathrm{ATGCTACGC}3\prime \left(\mathrm{DNA}\mathrm{sequence}\right)$

the mRNA formed from that sequence would be

$5\prime \mathrm{AUGCUACGC}3\prime \left(\mathrm{RNA}\mathrm{sequence}\right)$

remembering that U replaces T in RNA and that each letter represents a ribose mononucleotide.

The corresponding amino acids derived from the mRNA sequence would be

$N-\mathrm{Met}-\mathrm{Leu}-\mathrm{Arg}-C$

When A forms a base pair with T, there are two sets of hydrogen bonds involved, whereas the interaction between G and C involves three sets of hydrogen bonds ( Fig. 10.12 ), making the G–C interaction stronger than the A–T, or the A–U ( Fig. 10.11B ) interactions.

During cell division the genetic information has to be copied so that the daughter cell contains the same DNA as the parental cell. The two strands of DNA separate and DNA polymerase synthesizes a complementary strand (see the antiparallel strand above) so that for every A there is a T and for every G there is a C and vice versa following the rules of base pairing in the newly synthesized strand ( Fig. 10.33 ).

The synthesis of new strands of DNA ( DNA replication ) is the function of DNA polymerase III ( DNA pol III ). In human cells the rate of addition of nucleotides to the growing chain is about 100 per second. To attach a dNTP an RNA primer with a free 3′-hydroxyl is needed for the polymerase. The DNA polymerase has a proofreading capability that ensures the addition of the correct base. Through its inherent 3 ′, 5 ′ -exonuclease activity , the polymerase can remove a mistake. One of the subunits of the enzyme acts like a clamp to fasten the DNA polymerase to the DNA template.

There is a group of proteins, known as the primeosome , which contains a primase , function of which is to attach a small RNA primer to the single-stranded DNA. For DNA polymerase to start its synthetic activity, this primer provides a substitute 3′-hydroxyl group. At some point later on, the RNA primer is deleted by RNase H , leaving a gap that becomes filled by DNA pol I. When the RNA primer is removed, the unattached gap is filled in by DNA ligase that generates the formation of a phosphodiester bond at the 3′-end. Single-stranded binding proteins maintain the stability of the replication fork by helping helicase to make available the single-stranded template for DNA pol III. While the DNA polymerase moves easily on the leading strand in the direction of 5′–3′, the movement from the 3′-end toward the 5′-end of the lagging strand is more difficult. To continue in the 5′- to 3′-direction, the enzyme synthesizes stretches of the lagging strand in the 5′- to 3′-direction called Okazaki fragments . To complete the lagging strand, these stretches are filled by DNA pol I and the ligase.

At the beginning of the process, a topoisomerase called gyrase induces the unwinding of the double-stranded DNA. The processes of DNA replication are explained by Fig. 10.35A and B .

A-DNA proofreading and repair mechanism is part of the process that can recognize an improper base pair and replace it with the correct base, otherwise, there might be 1 incorrect base pair per 1000. DNA pol II is more involved in proofreading than the other polymerases. Because of this protective mechanism, the actual error rate in DNA synthesis is between one in 1 million and one in 1 billion. A repair enzyme can remove a lesion in one of the two DNA strands and the correct base pair can be added by reference to the undamaged strand. Corrections can occur as follows: DNA glyoxylase removes the altered base after the removal of the deoxypentose phosphate (base excision repair) or, in an alternate case, a small oligonucleotide stretch surrounding the damage is removed ( nucleotide excision ). In either case the gap is filled by DNA polymerase and DNA ligase. The adventitious conversion (by deamination) of cytosine to uracil is one case of a mismatched base pair. In this case, uracil could be removed and replaced with cytosine or, in another case, uracil might form a base pair with adenine to give rise to an unacceptable base pair . If the repair mechanism might, in error, excise the uracil and replace it with an adenine, a mutation would result. The experimentally proven model of DNA replication is called the semiconservative model . In this model, two strands of the parental DNA separate with each strand functioning as a template for the synthesis of a new complementary strand. The resulting first generation would be hybrid molecules, one strand from the parent and one new strand. In the second generation, there would result four double-stranded DNAs, two of which are hybrid molecules and two of which derive from the new DNA in the first generation as shown in Fig. 10.36 .

Mitochondrial DNA Synthesis

There are 37 genes within a circular DNA in human mitochondria. The inheritance of these genes is exclusively maternal (the lesser concentration of paternal mitochondria is possibly diluted out during the process of embryogenesis) and the mitochondrial genetic material is not replicated coordinately with nuclear DNA (although two proteins involved in mitochondrial DNA (mtDNA) replication are encoded by nuclear genes) . When a mitochondrion has grown to a point when it begins to divide by fission, replication of the DNA will begin, a process that is similar to bacterial DNA replication (fortifying the idea that mammalian mitochondria are derived from bacteria). It is theorized ( endosymbiotic hypothesis ) that during evolution some form of bacteria, possibly purple nonsulfur bacteria, survived the endocytotic process of some early cell type and eventually became incorporated into the cytoplasm as mitochondria, complementing the cytoplasmic metabolism with a more efficient oxidative metabolism. Thus the replication of mtDNA is similar to bacterial replication of its DNA. Mitochondrial genes do not code for all the enzymes and other proteins of the mitochondrion. This may have been the case long before the evolution of mammalian cells progressed and many of the genes in the early genome of the mitochondrion may have been lost, while some of the essential genes became relegated to the nuclear compartment. Of the mtDNA, 80% codes for functional mitochondrial proteins.

In mtDNA replication, both DNA strands are continuously synthesized as leading strands in the 5′- to 3′-direction. After the first strand begins to be synthesized, there is a pause until a signal is generated for completion of the strand. This pause causes the formation of loops (called D loops ) as shown in Fig. 10.37 that outlines the mechanism of mtDNA replication.

The human mitochondrion has 37 genes [encoding: 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and 13 polypeptides that are subunits of enzymes in the oxidative phosphorylation system]. Nuclear genes control mitochondrial replication. Two proteins in the mtDNA replication system are encoded by nuclear genes: these are a D-loop endoribonuclease and DNA primase . These proteins are presumably synthesized in the cytoplasm and transported into the mitochondria. DNA primase initiates mitochondrial replication at an origin.

When the heavy strand of mtDNA is transcribed, a transcript results that contains information for tRNA , rRNA , and mRNA on a polycistronic molecule and the full-length transcript is cut into the functional RNA units. The initiation of transcription requires three proteins: mitochondrial RNA polymerase, and four mitochondrial transcription factors , A, B1, and B2 (consisting to two factors). These proteins are assembled on the mitochondrial promoters to initiate transcription.

The synthesis of the second strand of DNA begins after the fork of the first strand synthesis passes through the origin of the second strand. The origins for left- and right-strand replication are separate for mtDNA but coincident for nuclear DNA. When synthesis has been completed, the products are two daughter circular double-stranded mtDNA molecules to be included in two daughter mitochondria. Some will be smooth circles and others will have D loops ( Fig. 10.37 ). A more detailed diagram outlining mtDNA replication is shown in Fig. 10.38 .

DNA Mutations and Damage

Mutations in human DNA can occur from a number of different sources, including radiation and exposure to toxic chemicals. Many carcinogens (some are DNA intercalating agents) are incorporated into the structure of DNA and cause mutations. Cosmic radiation (gamma radiation) rarely strikes the DNA molecule directly to cause a mutation. Mutations also can occur by viruses. Base deamination and depurination also can result in mutation. Adaptation from a very warm climate to a cold climate may require mutations in mtDNA affecting the usage of ATP, either primarily toward energy or the production of bodily warmth. These mutations can be passed to future generations, in the case of climatic change, adapting the human from the warmth of Africa to the colder climbs of Europe and North America, for example.

Some alterations in DNA can be corrected by repair mechanisms, although others are irreversible. As already discussed, DNA polymerase has a proofreading system that reduces mistakes in replication. Exposure to ultraviolet light can cause the appearance of a thymine dimer formed between two adjacent thymine bases in the same strand of DNA. This lesion can be repaired by actions of a nuclease, DNA polymerase, and ligase as shown in Fig. 10.39 .

Xeroderma pigmentosum is a rare autosomal recessive genetic disease. Such individuals are extremely sensitive to UV radiation and often develop basal cell carcinomas at an early age. Although there are several forms of xeroderma pigmentosum , most of the variants of this disease involve deficiencies in the nucleotide excision repair mechanism . Resulting DNA lesions are mainly the formation of thymine dimers and other photoproducts. This disease can result in continuing changes in the eyes and skin to progressive neurological degeneration. Xeroderma pigmentosum is six times more prevalent in Japanese than in other groups. Carcinogens are chemicals that are either direct or indirect acting. Examples of the direct acting group (chemicals that interact directly with DNA) are β-propiolactone, ethyl methanesulfonate , nitrogen mustard , and methyl nitrosourea . Some indirect carcinogens (chemicals that must be metabolized in the body to forms that interact directly with DNA) are benzo(a)pyrene , dibenzanthracene , 2-napthylamine , dimethylnitrosamine , vinyl chloride , acetylaminofluorene , and aflatoxin B1 . The several types of DNA damage are listed in Table 10.2 .

Epigenetics

Every cell in the human body contains a genome that consists of about 20,000 genes. The totality of the genes is identical in every cell; however, cells and tissues become differentiated to perform specific functions in the body. These changes occur through the actions of numerous small molecules that generate specific signals during development . In this way, some genes are expressed in some cells but are silenced in others. The different array of genes that are active and those that are silent define the differentiated cells. These arrays are then carried through to subsequent germline generations.

Epigenetics describes the conditions in which the mature body is exposed to different environments, external or internal, and these become imprinted in DNA. The scope of these environmental changes is enormous and effects that are carried through from one generation to the next and beyond can reflect effects of the external climate or even effects upon internal psychology. For example, humans raised in a very hot and bright climate would have dark skin where the expression of melanin protects the skin from excessive radiation. After migrating to a northerly cold and relatively dark environment, a lighter colored skin develops owing to a decrease in the production of melanin, an alteration that would be carried through to subsequent generations. Such lasting changes result from DNA methylation that cause silencing of gene expression, in this case, the expression of melanin.

In epigenetic methylation the bases that can be methylated are adenosine and, preponderantly, cytosine. The CpG base to be methylated is extended out of the DNA double helix into the active site of DNA methyl transferase where it can accept the active methyl group from S -adenosylmethionine (SAM). The enzymatic system of the methylation of cytosine is shown in Fig. 10.40 . The methyl group of methionine in the form of SAM is transferred to the 5 position on cytosine (see page 260 for numbering of the cytosine ring). The product of SAM after the transfer of the methyl group is S -adenosyl homocysteine which, through hydrolytic action, generates homocysteine that can be reutilized to reform SAM. The cofactor for the methylation of homocysteine to form methionine is THF . The overall effect of cytosine methylation on gene expression at the level of chromatin is shown in Fig. 10.41 .

Restriction Enzymes

Nucleases that can cut DNA at specific sites have been discovered in bacteria and the enzymes are commercially available. These enzymes are called restriction endonucleases or simply, restriction enzymes . Endonucleases cut DNA in the interior of the molecule, while exonucleases cut DNA at the ends of the molecule. Restriction enzymes bind to the double-stranded DNA and then travel along the molecule until the signal sequence (substrate) is encountered where the enzyme stops traveling and performs its nuclease function on both strands. The target site, recognized by the enzyme, usually consists of four, six, or eight bases in sequence from the 5′ to 3′ direction. The restriction enzymes are named for the organisms from which they are derived using an abbreviated form (e.g., Eco R1 from Escherichia coli ). As an example, Eco R1 catalyzes the cleavage of DNA between the G and the A of the sequence 5′-GAATTC-3′ to generate the products: 5′…G and AATTC…3′, as shown in Fig. 10.42 along with the descriptions of three other restriction enzymes.

In Fig. 10.42 , Hae III and Sma I produce blunt-ended products, that is, products, ends of which align. Eco R1 and Hind III produce “sticky ends” from the cleavage in that one set of the bases overlaps with the other. Fig. 10.43 shows atomic models of Eco R1 bound to double-stranded DNA and the release of the cleaved DNA strands.

Sticky ends are available for the insertion of foreign or specifically designed DNA; these can be engineered to have the complementary sticky end sequences. TTAA is the overhanging end in the case of Eco R1 and this can be glued to another DNA with an overhang of AATT, because TT would form base pairs with AA and AA would form base pairs with TT to appear like

$\begin{array}{ccc}\mathrm{DNA}1& \mathrm{TTAA}+\mathrm{AATT}& \mathrm{DNA}2\leftrightharpoons \\ & \mathrm{TTAA}& \mathrm{DNA}1\\ & \mathrm{AATT}& \mathrm{DNA}2\end{array}$

where hydrogen bonds would form between T:A, T:A, A:T, and A:T. In a given span of a DNA, there may be a number of restriction sites for a specific restriction enzyme . For example, in a 50-kb DNA, there might be a half a dozen Eco R1 target sites . Hydrolysis by Eco R1 would create a signature of the products.

There are hundreds of restriction enzymes with widely varying specificities, each one having its own target sequence as shown in Table 10.3 .

Table 10.3

A List of Many Restriction Endonucleases With Their Recognition Sites in DNA.

Source: Data in this table were taken originally from http://www.bioscience.org .

Enzyme	Recognition Site	Enzyme	Recognition Site	Enzyme	Recognition Site
Aat II	GACGI▾C	Cla I	AT▾CGAT	Nde I	CA▾TATG
AccI	GT▾|A/T||T/C|AC	Csp I	CG▾G(A/T)CCG	NgoM I	G▾CCGGC
AccIII	T▾CCGGA	Csp 45 I	TT▾CGAA	Nhe I	G▾CTAGC
Acc65 I	G▾GTACC	Dde I	C▾TNAG	Not I	GC▾GGCCGC
AccB7 I	CCANNNN▾NTGG	Dpn I	G me A▾TC	Nru I	TCG▾CGA
Acyl	C[A/G]▾CG[T/C]C	Dra I	TTT▾AAA	Nsi I	ATGCA▾T
Age I	A▾CCGGT	EclHK I	GACNNN▾NNGTC	Pst I	CTGCA▾G
Alu I	AG▾CT	Eco47 III	ACG▾GCT	Pvu I	CGAT▾CG
A/w26 I	G▾TCTC(1/5)	Eco52 I	C▾GGCCG	Rvu II	CAG▾CTG
A/w44I	G▾TGCAC	Eco72 I	CAC▾GTG	Rsa I	GT▾AC
Apa I	GGGCC▾	Eco I CR I	GAG▾CJC	Sac I	GAGGCT▾C
Ava I	C▾(T/C)CG|A/G|G	Eco RI	G▾AATTC	Sac II	CCGC▾GG
Ava II	G▾G(A/T)CC	Eco RV	GAT▾ATC	Sal I	G▾TCGAC
Ba/I	TGG▾CCA	Fok I	GGATG|9/13|	Sau3A I	▾GATC
BamH I	G▾GATCC	Hae II	(A/G)GCGC▾(TC)	Sau96 I	G▾GNCC
Ban I	G▾G(T/C)|A/G)CC	Hae III	GG▾CC	Sca I	ACT▾ACT
Ban II	G(A/G)GC(T/C)▾	Hha I	GCG▾C	Sfi I	GGCCNNNN▾NGCCC
Bbu I	GCATG▾C	Hinc II	GT|T/C|▾[A/G]AC	Sgf I	GCGAT▾CGC
Bcl I	T▾GATCA	Hind III	A▾AGCTT	Sin I	G▾G[A/T]CC
Bgl I	GCCNNNN▾NGGC	Hinf I	G▾ANTC	Sma I	CCC▾GGG
Bgl II	A▾GATCT	Hpa I	GTT▾AAC	SnaB I	TAC▾GTA
BsaM I	GATTGCN▾	Hpa II	C▾CGG	Spe I	A▾CTAGT
BsaO I	CG(A/G)|T/C|▾CG	Hsp92 I	G[A/G]▾CG|T/C|C	Sph I	GCATG▾C
Bsp1286 I	G(G/A/T)GC(C/A/T)▾C	Hsp92 II	CATG▾	Ssp I	AAT▾ATT
BsrBR I	GATNN▾NNATC	I-Ppo I	CTCTCTTAA▾GGTAGC	Stu I	AGG▾CCT
BsrS I	ACTGGN▾	Kpn I	GGTAC▾C	Sty I	C▾C[A/T][T/A]GG
BssH II	G▾CGCGC	Mbo I	▾GATC	Taq I	T▾CGA
Bst71 I	GCAGC(8/12)	Mbo II	GAAGA[8/7]	Tru9 I	T▾TAA
Bst98 I	C▾TTAAG	Mlu I	A▾CGCGT	Tthill I	GACN▾ NNGTC
Bst E II	G▾GTNACC	Msp I	C▾CGG	Vsp I	A▾TAAT
Bst O I	CC▾[A/T]GG	MspA I	C(A/C)G▾(G/T)G	Xba I	T▾CTAGA
Bst XI	CCANNNNN▾NTGG	Nac I	GCC▾GGC	Xho I	C▾TCGAG
Bst ZI	C▾GGCCG	Nar	GG▾CGCC	Xho II	[A/G]▾GATC[T/C]
Bsu36 I	CC▾TNAGG	Nci I	CC▾(G/C)GG	Xma I	C▾CCGGG
Cfo I	GCG▾C	Nco I	C▾CATGG	Xmn I	GAANN▾NNTTC

CAG , Chicken β-actin/globin.

Restriction enzymes are useful to isolate a specific fragment from linear or circular DNA. A DNA molecule can be characterized by its array of restriction sites. The products of digestion of DNA with restriction endonucleases can be separated by sizing gel electrophoresis . After electrophoresis the gel can be stained with ethidium bromide that binds to the fragments and the bound complexes become visible (yellow-green fluorescence) after exposure to ultraviolet light. In this way, restriction maps are developed; this is especially useful for vectors that are used to carry a specific DNA of interest. A restriction map of the vector pBR322 is shown in Fig. 10.44 .

The insertion of a foreign DNA into a plasmid by use of an Eco R1 restriction site is shown in Fig. 10.45 .

This is known as recombinant DNA . This can also be generated using restriction enzyme cleavage that results in blunt ends rather than overhanging sticky ends . When blunt ends are the products, synthetic sticky ends can be added. Using terminal transferase , poly d(G) can be added to 3′ blunt ends of the vector, while poly d(C) can be added to the 3′-ends of the foreign DNA to be inserted. The new chimeric (hybrid) DNA cloning vector can be amplified in host cells ( Fig. 10.46 ).

Specific human proteins can be expressed in relatively large quantities for therapeutic use. As the sequences expressed are human, little, if any, antigenic response should be encountered after administration. By the usage of restriction enzymes and different cloning vectors, the complete genome of an organism can be incorporated into a vector. As the therapeutic use of substances from animal tissues (e.g., hormones isolated from animal brains, such as growth hormone), in some cases, cause an antigenic reaction or can carry the danger of disease (e.g., prion disease via the scrapie protein transmitted from brains of diseased animals), the use of synthetically derived human sequences would be desirable. A general scheme describing the steps in gene cloning is shown in Fig. 10.47 . As an example of the preparation of a human protein for clinical use, cloning and reproducing the human insulin gene is shown in Fig. 10.48 .

A collection of recombinant clones constitutes a genomic library that contains the total DNA from a specific cell. Genomic DNA can be cut by restriction endonucleases so that large pieces of DNA fragments are obtained increasing the chances that the full sequences of individual genes are conserved. Certain vectors are useful in this respect because they can incorporate large fragments of DNA. Among the vectors that accept large DNAs are YAC ( yeast artificial chromosome ), BAC ( bacterial artificial chromosome ), and P1 ( bacteriophage-derived vector ). YAC can carry DNAs from 100- to 3000-kb pairs and can be propagated in E. coli . BAC can incorporate DNAs from 100- to 300-kb pairs and can be propagated in bacteria. P1 can incorporate DNAs from 130- to 150-kb pairs. A vector that contains a gene for a protein that can be expressed in active form is an expression vector . New technologies increase the efficiency and decrease the cost of producing relatively large amounts of human proteins. Although some regions of the human genome are unstable and difficult to clone, a human artificial episomal chromosomal system has been developed that appears to be a good candidate for storage and expression.

Probing Libraries for Specific Genes

A complementary DNA (cDNA) probe can be generated from a specific mRNA. The mRNA, encoding a specific protein, is a template. By the action of reverse transcriptase and DNA polymerase , a cDNA is formed that can be used as a probe to hybridize with a specific gene sequence ( Fig. 10.49 ).

Generally, the cDNA probe will be labeled, more recently, with a fluorescent tag that does not interfere with the hybridization reaction. Such a cDNA can be used to probe a library of cDNAs for a complementary sequence, either to find a longer sequence containing more information or to search out a full coding region of the gene.