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ScienceWeek
MOLECULAR BIOLOGY: ON DNA POLYMERASES
The following points are made by O. Fleck and P. Schär (Current Biology 2004 14:R389):
1) Before a cell can divide, it must ensure that its daughters will receive an identical copy of their genome so that the chain of inheritance is maintained. Genome duplication is accomplished by efficient DNA replication machines which, in a human cell, manage to synthesize billions of nucleotides within a few hours and with a fidelity of less than one error every billion steps. At the heart of these machines lie the DNA polymerases.
2) Evolution has produced a number of different types of DNA polymerase, but they all have a similar overall three-dimensional shape that has been likened to a right hand, with palm, finger and thumb domains [1]. Polymerases of the A and B families, such as polymerase d, replicate the bulk of genomic DNA during the cell cycle and have been streamlined for processivity and accuracy. These enzymes fit the DNA substrate tightly into their active site, where the replicating base pair is enclosed by the finger domain [2-4]. The mobility of the finger domain underlies a so-called "induced-fit" mechanism for checking the fidelity of replication: only when an incoming nucleotide forms a perfect Watson-Crick pair with the template base can the fingers close and induce an active conformation of the polymerase. If, nevertheless, an erroneous nucleotide happens to be incorporated, the polymerase responds with conformational distortions of its active center. These induce replication pausing and translocation of the primer terminus towards the intrinsic "proofreading" exonuclease activity, which removes the mispairing base so that synthesis can resume [5].
3) In any cell, however, the genomic DNA has damage that is incompatible with the formation of smooth Watson-Crick base pairs. This applies particularly to abasic sites, which arise as a consequence of spontaneous base hydrolysis or via the excision of damaged bases by DNA glycosylases. Estimates put the number of such events at 10,000 per mammalian cell per day, most of which will be repaired by base excision repair pathways. Some, however, will persist and impede DNA replication by stalling the replicative DNA polymerases. The processivity and fidelity of these enzymes seems of little help when it comes to traversing a truly non-instructive lesion. What, after all, is the purpose of fidelity when facing a lost identity?
4) Bypassing such lesions requires specialists that provide tolerance to aberrant DNA structures and creativity in interpreting non-instructive lesions. For this reason, evolution has created the Y-family of DNA polymerases, the most prominent member of which is polymerase eta, defective in humans with xeroderma pigmentosum variant (XP-V). Y-polymerases are characterized by a distributive mode of action, a high error rate when replicating non-damaged DNA and an ability to synthesize DNA across and past a variety of template lesions. In addition to the universal thumb, palm and finger domains, they have a unique carboxy-terminal domain, their "little finger". The palm and finger domains form a spacious active site, accommodating the DNA substrate through a few, largely non-specific contacts. The thumb and little finger domains grip the duplex portion of the DNA from the minor and major groove sides, respectively. Together, the open active site geometry and a relaxed dependency on specific DNA interactions result in the ability of these polymerases to tolerate DNA substrate distortions at or beyond the active site.
References (abridged):
1 Hübscher, U., Maga, G., and Spadari, S. (2002). Eukaryotic DNA polymerases. Annu. Rev. Biochem. 71, 133-163
2 Ollis, D.L., Brick, P., Hamlin, R., Xuong, N.G., and Steitz, T.A. (1985). Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP. Nature 313, 762-766
3 Doublie, S., Tabor, S., Long, A.M., Richardson, C.C., and Ellenberger, T. (1998). Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution. Nature 391, 251-258
4 Li, Y., Korolev, S., and Waksman, G. (1998). Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. EMBO J. 17, 7514-7525
5 Carver, T.E.Jr. , Hochstrasser, R.A., and Millar, D.P. (1994). Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I. Proc. Natl. Acad. Sci. USA 91, 10670-10674
Current Biology http://www.current-biology.com
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Related Material:
MISREGULATION OF MITOSIS AND HUMAN AGING
Notes by ScienceWeek:
The idea that human aging is controlled by genes is not the end but the beginning of research: Does the aging process involve one gene, or a fixed group of specific genes, or groups of various genes at different times and in different individuals?
The term "messenger RNA" refers to the ribonucleic acid molecule transcribed from DNA that carries the coded information specifying the sequence of amino acids in a protein. Since the various messenger RNAs present in any cell can be isolated and identified, the population of present messenger RNAs can be used to establish a profile of the active genes of that cell.
Fibroblasts are a type of connective tissue cell secreting structural proteins such as collagen, the proteins forming a matrix in which the fibroblasts become embedded. These cells can be easily obtained from skin, and they can be easily cultured outside the body.
Progeria (Hutchinson-Gilford disease; Hutchinson-Gilford syndrome; premature senility syndrome) is a condition of precocious aging, with onset at birth or early childhood, the condition characterized by growth retardation, a senile appearance with dry wrinkled skin, early occurrence of *atherosclerosis in blood vessels, and premature death (usually before the age of 20) due to coronary artery disease. The disease is apparently genetic, but the details of the etiology are not clear.
In this context, the term "phenotype" refers to the specific individuality of an organism as determined by the interaction during development between its genetic constitution (genotype) and the environment.
In this context, the term "mitosis" refers to the entire cell division phase of the cell cycle, with "cell cycle" referring to the entire life history of a single cell from mitosis to mitosis, including the sequence of intervening phases.
In this context, the term "postmitotic cells" refers to cells that normally do not undergo mitosis once they have fully differentiated (e.g., neurons and muscle cells).
The following points are made by D.H. Ly et al (Science 2000 287:2486):
1) The authors measured messenger RNA levels in actively dividing fibroblasts isolated from young, middle-age, and old-age humans and humans with progeria. Messenger RNA levels were analyzed with high-density *oligonucleotide arrays containing probes for more than 6000 known human genes.
2) The authors report their results suggest that an altered expression profile of genes involved in mitosis occurs with age, and that these changes result in increased rates of *somatic mutation, leading to numerical and structural *chromosome aberrations and mutations that manifest themselves as an aging phenotype.
3) The authors suggest that these chromosome pathologies, which begin to occur in dividing cells relatively early in life (but in the postreproductive stage), may lead to misregulation of key structural, signaling, and metabolic genes associated with the aging phenotype, such as the apparent misregulations characteristic of *osteoporosis, *Alzheimer's disease, *arthritis, etc. Misregulation of this sort is expected to increase in each round of cell division, and it may be propagated to other normal mitotic cells (e.g., *leukocytes, *epithelial cells, *glial cells, etc.) and postmitotic cells (e.g., neurons, muscles, etc.) through changes in the *extracellular matrix and oxidized fatty acid derivatives that affect signaling pathways. Aging, the authors suggest, may therefore occur gradually and in mosaic patterns, rather than as a uniform phenomenon characteristic of cancerous growth (which is clonal -- deriving from a single mutated progenitor cell).
4) The authors conclude: "Additional studies are required before we can understand the aging process in complex organisms, both in mitotic and postmitotic tissue, but the studies reported here highlight important mechanisms that may contribute to aging and age-related problems."
Science http://www.sciencemag.org
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Notes by ScienceWeek:
atherosclerosis: "Arteriosclerosis" is a generic term for several diseases in which the arterial wall becomes thickened and loses elasticity, and "atherosclerosis" is a form of arteriosclerosis characterized by patchy thickening (atheroma) in the subintimal layer (i.e., immediately below the innermost layer [intima]) of medium and large arteries, the thickening capable of reducing or obstructing blood flow.
oligonucleotide arrays: (DNA microarrays) DNA microarrays are chips containing hundreds or thousands of gene snippets laid out in precise arrays that provide rapid snapshots of the expression of whole suites of genes. The general method in microarray analysis is to a) isolate messenger RNAs (mRNAs) produced by a genome; b) convert mRNA into complementary DNA (cDNA); c) add a fluorescent tag to the cDNA for tracking purposes; d) wash a solution of tagged cDNAs over a DNA microarray chip. Each DNA snippet on the chip will bind the cDNA from the corresponding gene, and by measuring the fluorescences arrayed on the chip, the profile of gene expression is revealed.
somatic mutation: In general, a mutation occurring in non-germ cells, which means the mutation is not transmitted to the next generation of individuals (but is transmitted to the next generation of cells of that type).
chromosome: In cells with chromosomes, the chromosomes are the physical structures into which DNA is organized and on which genes are carried.
osteoporosis: A generalized progressive diminution of bone density (bone mass per unit volume) that causes skeletal weakness. The ratio of mineral to organic elements is unchanged. The major clinical manifestations of osteoporosis are bone fractures resulting from a reduction below the fracture threshold of the amount of bone available for mechanical support.
Alzheimer's disease: There are various forms of dementia produced by various causes. Alzheimer-type dementia (Alzheimer's disease) is apparently related to what appear to be specific cellular and histological degenerative processes, with loss of cells from several specific brain areas, the brain showing moderate to marked atrophy. Memory loss is the most prominent early symptom.
arthritis: In general, inflammation of a joint or a state characterized by inflammation of joints.
leukocytes: White blood cells, of which there are various types.
epithelial cells: In animals, "epithelial cells" compose the cell layers that form the interface between a tissue and the external environment, for example, the cells of the skin, the lining of the intestinal tract, and the lung airway passages.
glial cells: Cells of the central and peripheral nervous system that metabolically support neurons. Such cells also produce the multiple membrane layers called myelin and enfold certain nerve cell axons with it.
extracellular matrix: In general, the extracellular matrix is a layer consisting mainly of proteins and glycosaminoglycans that form a sheet underlying endothelial and epithelial cells. The molecular constituents of the matrix are secreted by cells in the vicinity. Endothelial cells are the cells that line blood vessels.
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