|
ScienceWeek
MOLECULAR BIOLOGY: ON THE MECHANISM OF DNA REPLICATION
The following points are made by Stephen D. Bell (Nature 2006 439:542):
1) DNA is replicated by unzipping the double helix to expose the bases that act as a template for copying the genetic material. Both strands of DNA serve as templates, and thus one double helix becomes two. Conceptually, this is a simple reaction, but the devil -- as so often -- is in the detail: the process is mediated by a multitude of proteins and turns out to be mechanically complex. New work [1-3] has made considerable headway in understanding the intricacies of replication.
2) One level of complexity in the replication reaction comes from the fact that DNA polymerase, the enzyme that synthesizes the new DNA, cannot begin a strand itself. Rather, it extends a short RNA "primer" that is already bound to the template. This means that an additional enzyme, a primase, is required to generate the primer to start the polymerase reaction[4,5].
3) A second complexity lies in the fact that the two strands of the DNA double helix are arranged antiparallel to one another; the opposite directions are termed 5' to 3' and 3' to 5' (from the positions of carbon atoms in the sugars that make up the DNA backbone). However, DNA polymerase can synthesize DNA in only one direction: 5' to 3'. So the 3' to 5' template strand -- the "leading" strand -- can readily be replicated, but how is the other, "lagging" strand copied? This dilemma was resolved by the discovery that the lagging strand is replicated discontinuously. It is synthesized in short pieces, called Okazaki fragments, that are then joined together -- essentially, the polymerase takes two "steps" forward and then synthesizes one back. The difference in the synthesis of the two strands means that, in principle, the leading strand requires only a single priming event, whereas the lagging strand needs a new primer for each Okazaki fragment. And all these events must somehow be coordinated to produce two daughter strands at roughly the same rate.
4) One of the outstanding conundrums concerning replication is how the process deals with broken or damaged DNA, for example that generated by ultraviolet radiation, particularly at the leading strand. Replicating the damage could obviously be harmful for the daughter cells. So does the replication machinery just stall and wait for the damage to be fixed, or is there some way to sidestep the damage and continue without copying the damaged DNA? Heller and Marians[1] addressed this issue by biochemically mimicking a blockage of the leading-strand template. Their findings indicate that a new priming event can occur downstream of the lesion, so that the portion of the leading strand encompassing the lesion is not copied and the resulting DNA will be single-stranded. This would seem to contradict the prevalent theory that leading-strand synthesis is continuous. However, precisely this phenomenon is observed in bacterial cells, where ultraviolet irradiation can lead to single-stranded gaps on both leading and lagging strands.
References:
1. Heller, R. C. & Marians, K. J. Nature 439, 557-562 (2006)
2. Lee, J. -B. et al. Nature 439, 621-624 (2006)
3. Zenkin, N. , Naryshkina, T. , Kuznedelov, K. & Severinov, K. Nature 439, 617-620 (2006)
4. Kornberg, A. & Baker, T. A. DNA Replication 2nd edn (Freeman, New York, 1992)
5. Frick, D. N. & Richardson, C. C. Annu. Rev. Biochem. 70, 39-80 (2001)
Nature http://www.nature.com/nature
--------------------------------
Related Material:
MOLECULAR BIOLOGY: ON THE PACKING OF DNA IN NUCLEOSOMES
The following points are made by J.C. Eissenberg and S.C. Elgin (Nature 2005 438:1090):
1) Crack open any cell nucleus and look inside: you will see what look like beads on a string. The beads are nucleosomes, small protein complexes that help to package the DNA (the strings) into the cramped confines of the nucleus. In the past 15 years, nucleosomes have graduated in our understanding from being passive spools for DNA to full partners in the control of genetic information in the cell. Diverse chemical modifications of the histone proteins that form the nucleosome core can alter the expression of the associated genes[1]. These modifications make up what is known as the "histone code", and a major challenge in molecular biology is to decipher how they affect gene expression.
2) Two of the most common modifications are phosphorylation and methylation -- respectively the addition of a phosphate or a methyl group to the amino acids of which the histones are composed. Allis et al[2] previously proposed that reversible phosphorylation of the amino acids serine or threonine in the "tail" regions of histones could antagonize the binding of regulatory proteins to neighboring methylated lysine amino acids, creating a binary control switch. New work[3,4] presents data that strongly support this model and advances our understanding of how histone modification can control chromosome function.
3) Among the many histone modifications that have been described, methylation of lysines and phosphorylation of serines and threonines have attracted much attention. Phosphorylation of the serine at the tenth position in the tails of histone H3 (H3S10) occurs during cell division in eukaryotic (higher) cells. Once the cells have replicated their DNA and begin to prepare for division, nearly all of the histone H3 in the nucleus seems to be phosphorylated at this site. Phosphorylation of H3S10 also occurs at other stages in the cell cycle, but only at discrete chromosomal sites that are associated with gene expression.
4) The methylation of lysines is more complex. Methylation at lysine 9 of histone H3 (H3K9) is found mainly in the heterochromatin -- the dense, mostly inactive regions of the genome. Methylation at lysine 4 of histone H3 (H3K4), by contrast, is associated with active genes. The different outcomes of lysine methylation result from the fact that each modification creates a binding target for a distinct protein. Heterochromatin protein 1 (HP1), which promotes heterochromatin formation (and the consequent gene silencing), recognizes and binds to methylated H3K9 using a region called the chromodomain[5]. CHD1, an enzyme that may destabilize nucleosomes and expose the DNA for gene expression, recognizes and binds to methylated H3K4 through two tandem chromodomains.
5) Fischle et al[3] and Hirota et al[4] examined cells progressing into the metaphase stage of cell division, where the chromosomes become very tightly packed, or "condensed", to facilitate their separation into the future daughter cells. Both groups discovered that these cells accumulated histone H3 that is both methylated at lysine 9 and phosphorylated at the neighboring serine 10. Using antibodies specific for the doubly modified H3, the authors found that this dual modification occurred specifically in heterochromatic regions of chromosomes.
References (abridged):
1. Khorisanizadeh, S. Cell 116, 259-272 (2004)
2. Fischle, W. , Wang, Y. & Allis, C. D. Nature 425, 475-479 (2003)
3. Fischle, W. et al. Nature 438, 1116-1122 (2005)
4. Hirota, T. , Lipp, J. J. , Toh, B. -H. & Peters, J. -M. Nature 438, 1176-1180 (2005)
5. Jacobs, S. A. & Khorisanizadeh, S. Science 295, 2080-2083 (2002)
Nature http://www.nature.com/nature
--------------------------------
Related Material:
GENOME BIOLOGY: STRUCTURE OF DNA IN THE NUCLEOSOME
The following points are made by T.J. Richmond and C.A. Davey (Nature 2003 423:145):
1) DNA in eukaryotic cells is packaged repetitively into nucleosomes by means of extensive association with histone proteins. The hierarchical chromatin structure formed is the genomic substrate relevant to the vital processes of DNA replication, recombination, transcription, repair, and chromosome segregation, and to the pathological progression of cancer and viral disease. Although nucleosomal organization of DNA is essentially ubiquitous throughout genomes and generally repressive to gene expression, it also contributes to gene transcription in a gene-specific manner, suggesting that nucleosome positioning in gene promoter regions is important for genuine gene regulation in vivo. The question therefore arises of how chromatin structure, in which DNA is normally highly compacted, permits site-specific access to regulatory factors and more extensive exposure to the transcription apparatus.
2) The answer is likely to require a knowledge of DNA conformation in the nucleosome core. The core comprises 147 base pairs (bp) of DNA and the histone octamer; compared with the nucleosome, it lacks only 10 90 bp of linker DNA envisaged to be naked or bound to histone H1. The histone-fold domains of the octamer organize the central 129 of 147 bp in 1.59 left-handed superhelical turns with a diameter only fourfold that of the double helix. The relatively straight 9-bp terminal segments contribute little to the curvature of the complete 1.67-turn superhelix. So far, the site-specific regulatory factors that have been discovered bind the linker or terminal regions of the intact nucleosome. The lack of binding to the central region of the superhelix might simply be a consequence of bending the double helix or, additionally, of unusual DNA conformations induced by histone binding. At least one protein, HIV-1 integrase, does prefer DNA bent around the nucleosome in contrast to naked DNA.
3) The initiation of DNA-dependent nuclear processes in the context of chromatin implies that nucleosome position is biased by the DNA sequence to facilitate access by initiation factors. Numerous examples of positioned nucleosomes in gene promoter regions have been described both in vivo and in vitro. Preferential positioning could place factor-binding sequences in nucleosome linker or terminal region DNA. Furthermore, nucleosomes are intrinsically mobile and yield access to their DNA in vitro, allowing even RNA polymerase to transcribe nucleosomal DNA without causing dissociation of the histone octamer. In vivo, energy-dependent chromatin remodeling factors, targeted by gene regulatory proteins and acting directly on the nucleosome core, augment nucleosome mobility. Their mechanism of action most probably derives from the innate ability of nucleosomes to "slide" along DNA without releasing it.
4) In summary: The 1.9-angstrom-resolution crystal structure of the nucleosome core particle containing 147 DNA base pairs reveals the conformation of nucleosomal DNA with unprecedented accuracy. The DNA structure is remarkably different from that in oligonucleotides and non-histone protein DNA complexes. The DNA base-pair-step geometry has, overall, twice the curvature necessary to accommodate the DNA superhelical path in the nucleosome. DNA segments bent into the minor groove are either kinked or alternately shifted. The unusual DNA conformational parameters induced by the binding of histone protein have implications for sequence-dependent protein recognition and nucleosome positioning and mobility. Comparison of the 147-base-pair structure with two 146-base-pair structures reveals alterations in DNA twist that are evidently common in bulk chromatin, and which are of probable importance for chromatin fiber formation and chromatin remodeling.
Nature http://www.nature.com/nature
ScienceWeek http://scienceweek.com
|