Chromosome Structure and Function

Chromosome structure and function Organization of Chromosomes Chromosomes contain genetic information in double-stranded DNA molecules A cell's genome - the total DNA content of a cell - is divided among one or more chromosomes that organize, store, and transmit information that guides the production of proteins and RNA molecules Interspersed between the genes are regions of DNA known as intergenic DNA that do not, to our knowledge, contain genes The genes of less complex, single-celled organisms, such as bacteria or budding yeast, are located fairly close to one another in the chromosome, with little intergenic DNA. By contrast, mammals have a lower density of genes within their chromosomes and more intergenic DNA Each species has a characteristic set of chromosomes Every organism maintains a fixed number of chromosome in each cell. The number of chromosomes per cell, along with the size of each chromosome, is a defining characteristic of each species Most eukaryote cells have multiple chromosomes within a membrane-bound compartment known as the nucleus. Human cells contain 46 chromosomes in the nucleus, half of which are inherited from the mother and half from the father Another defining characteristic of each type of cell is the number of sets of identical chromosomes it contains, a characteristic described by the term ploidy The copy of each chromosome inherited from the mother is homologous to the copy inherited from the father, meaning that it contains the same genes and gene order but may contain different versions of each gene; these different versions are called alleles Other classes of DNA can be found within cells Mitochondria, the membrane-enclosed organelles in eukaryote cells that are the primary source of cellular adenosine triphosphate (ATP), contain a single chromosome that is usually circular The presence of distinct mitochondrial DNA is one of the reasons that mitochondria are thought to have arisen from independently living bacteria that were ingested by another single-celled organism, within which the bacteria developed a symbiotic relationship Chloroplasts, the plant organelles where photosynthesis occurs, also have their own circular chromosome and are thought to have originated from blue-green algae, incorporated into another cell by a similar, but independent, event during evolution In addition to their main genomic DNA, bacterial cells can contain double-stranded circular DNA molecules known as plasmids, which contain a small number of genes The Cell Cycle and Chromosome Dynamics Chromosomes are duplicated and segregated to daughter cells during the cell cycle The faithful transmission of genetic information from one generation to the next depends on a cell’s ability to make copies of each chromosome and then distribute the complete set of chromosomes to the two daughter cells The incorrect segregation of chromosomes - for example, where one daughter cell receives an extra chromosome and the other cell receives none - may have a serious, negative impact on the viability of either or both daughter cells Before a cell can divide, it must replicate its DNA and segregate the duplicated chromosomes equally between what will become the two daughter cells The eukaryotic cell cycle is divided into four distinct phases G1: gap phase 1, cell growth occurs until cells attain a minimum size that is required to progress to the next phase S phase: DNA synthesis, DNA is copied, thereby duplicating all of the chromosomes. The two identical copies of each chromosome, called sister chromatids, remain physically associated with one another G2: gap phase 2, during which the cell prepares for the final phase M phase: mitosis, sister chromatids are separated and a complete set of chromosomes is delivered to each pole of the cell Mitosis is followed by cytokinesis, which completes the division of the mother cell into two daughter cells, each containing the same number of chromosomes As the cell enters M phase, the nuclear membrane is disassembled and sister chromatids condense into compact structures that remain bound together, which allows them to segregate without becoming entangled The centromeres are specialized regions of the chromosome that bind microtubules emanating from centrosomes, cellular structures that nucleate or establish the formation of microtubules and therefore define each pole of the mitotic spindle Chromosomes have a distinctive appearance at mitosis The most familiar picture of human chromosomes comes from a karyotype, a display of the chromosome set of an individual, lined up from largest to smallest The chromosomes typically seen in a karyotype are taken from microscopic images of metaphase chromosomes, when the sister chromatids are maximally condensed but have not yet separated Packaging chromosomal DNA DNA condensation occurs in multiple steps The first level of DNA packaging in all organisms is the binding of small, basic (positively charged) proteins directly to the DNA along its length. The many positively charged side chains on these proteins help to counterbalance the negatively charged DNA The compacted bacterial chromosome is called the nucleoid. The DHA within this DNA-protein complex becomes supercoiled as a result of its packaging - that is, it is wrapped around itself like a twisted rubber band and thus is much more compact than non-supercoiled DNA Eukaryotic DNA and histones are packaged into nucleosomes In eukaryotes, the basic DNA-binding proteins that package DNA into chromatin are called histones. There are four core histone proteins, H2A, H2B, H3, and H4, which evolved early in the history of eukaryotes All four of the core histones have a common protein fold, known as the histone fold, and are rich in lysine and arginine residues. The side chains of the amino acids lysine and arginine carry positive charges, which help to counterbalance the many negative charges on the phosphate groups in the DNA backbone Nucleosome: basic unit of DNA packaging in eukaryotes, consisting of a protein core composed of a histone octamer, with DNA wrapped around the outside The DNA winds around the histone octamer in a left-handed manner. The direction of this winding is important; when a histone octamer is stripped away from the DNA, it leaves behind negatively supercoiled DNA Each core histone protein has an N-terminal "tail" that extends away from the nucleosome core between the coils of DNA. These tails are stretches of around 30 amino acids that do not adopt a defined structure Nucleosomes form preferentially at certain DNA sequences The DNA sequence at the sharp bends generally consists of pyrimidine-purine base steps (the sequence thymine/adenine (TA) or cytosine/guanine (CG) as viewed along one strand), which are more highly bendable than other base steps A favored nucleosome binding region's sequence would contain AT-rich sequences alternating with stiffer GC-rich sequences, spaced roughly half a helical turn apart from one another Chromatin is organized into 30-nm filaments and higher-order structures This level of packaging arises from the way that histone octamers associate with doublestranded DNA to form nucleosomes, which are separated from one another by stretches of DNA to form a fiber that appears in electron micrographs like "beads on a string" Histone H1 binds to the linker DNA that connects successive nucleosomes, facilitating the compaction of chromatin. Histone H1 is therefore known as a linker histone Variation in Chromatin Structure Interphase chromatin is not uniformly packaged During interphase, chromosomes are in an extended, relatively decondensed form and many genes are actively transcribed. However, within the relatively decondensed chromosomes, we find significant variations in structure Euchromatin: active chromatin. DNA that is packaged as euchromatin is accessible to many DNA binding proteins that are involved in transcription, replication, recombination, and repair Heterochromatin: tightly packed chromatin that is less accessible to nuclease digestion than euchromatin and is associated with different chromosomal proteins The relative resistance of heterochromatic DNA to digestion is often interpreted as indicating that heterochromatin is more condensed than euchromatin, although it may also reflect differences in the ability of nucleases to interact with DNA bound by specific heterochromatin proteins Genes that are actively transcribed typically lie in regions of euchromatin, which is relatively decondensed. This open structure presumably allows ready access to DNA by the enzymes and proteins that must bind to it for transcription, repair, and recombination to occur Beyond the general occurrence of heterochromatin described earlier, certain specific regions of chromosomes also form heterochromatin in a characteristic way. These regions include the DNA surrounding the centromeres, which is the segment of the chromosome to which the spindle attaches for segregation, and at telomeres The structural state of chromatin affects all processes directed by DNA Telomeres are often packaged into heterochromatin. If a gene that is active in its normal location in , euchromatin is artificially placed adjacent to a telomere, the gene becomes silenced Differences in chromatin structure can also control which parts of the chromosomes are able to undergo recombination, a process by which a strand of DNA is broken and then joined to a different DNA molecule Covalent Modifications of Histones Modification of histones affects chromatin structure and function Histone proteins that package DNA into chromatin are subject to a variety of post-translational modifications. These modifications, which add chemical groups or small proteins to histone side chains, serve as important signals in regulating virtually all processes that involve interactions with chromatin Post-translational modifications occur both in the histone tails and in the globular core of histone proteins. Once modified, these sites are thought to recruit specific proteins to chromatin, which then affect chromatin structure and function An important feature of histone modifications is that the major ones are reversible. There are specialized enzymes that add particular chemical modifications to histone side chains, and other types of enzymes that remove the modification Histone acetylation is associated with active chromatin However, the relative levels of acetylation vary substantially; actively transcribed regions in euchromatin contain highly acetylated histones, whereas heterochromatin, which contains few transcribed genes, has very low levels of acetylation Enzymes called histone acetyltransferases (HATs) covalently attach acetyl groups to lysine side chains, while histone deacetylases (HDACs) remove the acetyl groups Bromodomain: protein domain that binds to acetylated lysine side chains - usually specific positions on a histone protein Histone methylation can be associated with either active or inactive chromatin Histones H3 and H4 can be methylated at both lysine and arginine residues. Up to three methyl groups can be covalently attached to a single lysine, while up to two can be attached to an arginine side chain Unlike acetylation, which is uniformly associated with active transcription, methylation is associated with either activation or repression of transcription. The precise effect of histone methylation depends upon which residue is methylated Histone phosphorylation and ubiquitination serve as additional regulatory signals Phosphorylated residues can be recognized by specialized protein domains, although the precise mechanism by which phosphorylation of histone exerts an effect remains to be elucidated The covalent attachment of ubiquitin of lysine side chains is the largest histone modification, since ubiquitin is a 76-amino acid protein, a notable contrast to the limited atoms added by each of the previously mentioned histone modifications Different patterns of histone modifications have distinct consequences The interdependence of some histone modifications, together with the large number of possible combinations of modifications within a single nucleosome, led to the suggestion that there is a histone code, in which unique combinations of modifications specify particular functional states of chromatin Nucleosome Remodeling Complexes ATP-dependent nucleosome remodeling alters the location of nucleosomes on DNA While the packaging of eukaryotic DNA into chromatin helps to organize the genetic material in the nucleus, it presents a potential barrier to different proteins and enzymes that must access the DNA This alteration is mediated by ATP dependent nucleosome remodeling complexes, which are multisubunit enzyme complexes that use the energy provided by ATP hydrolysis to change the positions of nucleosomes on DNA Nucleosome remodeling enzymes can either slide the histone octamer along DNA or remove the histone octamer entirely or a portion of it, and transfer it from one segment of DNA to another In addition, nucleosome remodeling complexes can increase the accessibility of DNA by introducing transient loops into the portion of DNA that is wrapped around a single histone core, thus increasing the accessibility of DNA to binding proteins The SWI/SNF complex, for example, contains 11 subunits, including an ATPase domain that catalyzes DNA-dependent ATP hydrolysis and a bromodomain that binds to acetylated lysine residues in histones or in other proteins associated with DNA It is also possible that a global distortion in the DNA, perhaps one that puts the DNA under torsional stress, could trigger repositioning of the nucleosome on the DNA Nucleosome remodeling complexes can be targeted to specific sites by interactions with other proteins Since nucleosome remodeling is typically required in precise locations on the DNA, the cell most have a mechanism for recruiting nucleosome remodeling complexes to specific sites on the DNA where they are needed In this way, chemical modification of histones can trigger the binding of nucleosome remodeling complexes, which, in turn, alter transcription or other processes involving access to the DNA DNA Methylation Methyltransferases add methyl groups to cytosine in DNA In bacteria, methyl groups can be added to adenine residues in DNA, and in both bacteria and eukaryotes, methyl groups can be added to cytosine residues to generate 5-methyl cytosine Methylated cytosines are chemically less stable than unmethylated cytosines and undergo rare deamination. This deamination of methyl cytosine generates a thymine in the DNA, and thus a methylated CpG dinucleotide becomes TpG This residue change can lead to a permanent change in the DNA base sequence because the single nucleotide mismatch may not always be recognized by the DNA repair machinery prior to replication Base flipping is used to give access to bases to be methylated The stable structure of DNA makes enzyme-driven base modification reactions, such as cytosine methylation, difficult. The bases are tightly packed inside the double helix, which is stabilized by base-pairing and base-stacking During base flipping, the cytosine base to be modified is flipped completely out of the DNA helix and an amino acid side chain from the enzyme is inserted in its place to stack temporarily with the adjacent nucleotides Bacteria use methylation to distinguish newly synthesized DNA and to mark and protect their own DNA While both strands of DNA are methylated in the growing cell, only the parental strand remains methylated immediately after DNA replication. We say that such a half-methylated site on a chromosome is hemi-methylated Likewise, DNA repair proteins search for nearby methylated adenines to identify the parental strand so that they can replace mismatched, incorrect bases in the newly synthesized strand using the correctly identified parental strand as the template Bacteria have specific endonucleases called restriction enzymes that cut DNA at sites containing particular recognition sequences; these enzymes are used to destroy foreign PNA that gets into the cell DNA methylation is associated with gene silencing in plants and mammals In other words, the presence of methylated cytosines along a DNA strand is interpreted by the cell to signal that DNA that is modified in this way should not be transcribed An important feature of this form of transcription repression is that the methylated - and hence silenced - state can be passed on to daughter cells, thereby maintaining the silenced state over many generations of cell division Certain protein complexes bind directly to DNA that contains methylated cytosine via specialized DNA methyl-binding domains. Proteins with these domains are often subunits of larger complexes, which are thus directed to methylated DNA DNA methylation is involved in X chromosome inactivation and genomic imprinting in mammals If both X chromosomes in a female were transcriptionally active, the female would express double the amount of gene product relative to the male, because the female has two copies of the gene being transcribed, rather than just the one X inactivation helps to correct the imbalance by switching Off one of the female's X chromosomes such that both sexes have the same number of X chromosomes in an active state The inactive X bears an the hallmarks of mammalian heterochromatin - DNA is heaving methylated, histones are modified as in heterochromatin, and most regions are completely inactive DNA methylation is also linked to a form of gene inactivation called imprinting, which is the silencing of one copy of a gene on either the maternal or the paternal chromosome, while the homologous genes on the other chromosome are expressed Elements Required for Chromosome Function All chromosomes contain specialized regions that direct essential chromosomal functions Chromosomes contain special features that allow them to be duplicated during DNA replication and to ensure that the resulting copies of chromosomal DNA are segregated into the two daughter cells at cell division Chromosome segregation in some bacterial species is directed by sequences near the replication origin. In eukaryotes, the centromere directs chromosome segregation Telomere: specialized structure at the end of a eukaryotic chromosome that enables the DNA to be fully replicated and also maintains the integrity of the chromosome DNA replication initiates at specific chromosomal sites termed origins For DNA replication to begin, the two strands of the DNA double helix must be unwound and separated to allow the replication machinery access to the DNA. Origins of replication (ori) are the sites at which this unwinding occurs The Centromere The centromere is a region of DNA necessary for chromosome segregation In most species, each chromosome has a single centromere, which is surrounded by heterochromatin and whose structure is stably inherited from one in generation to the next Kinetochore: a complex of proteins that attaches to the centromere and mediates the attachment of the chromosome to the spindle during mitosis During mitosis, kinetochores of sister chromosomes attach to microtubules emanating from opposite spindle poles. This attachment allows the spindle to pull sister chromatids apart and distribute them to the daughter cells Centromeres bind a histone H3 variant called CENP-A Despite dramatic sequence differences, centromeres have a defined region in which some nucleosomes contain a histone H3 variant, known as CENP-A. CENP-A shows specificity for centromere regions, and particularly the AT-rich domains The presence of CENP-A is crucial for centromere function; cells that lack CENP-A fail to recruit most known kinetochore components to the kinetochore CENP-A protein levels are carefully regulated, as abnormally high CENP-A protein levels can lead to binding at non-centromeric sites and can disrupt kinetochore function Chromatin structure defines centromere function While the function of many chromosomal elements depends on their DNA sequence, surprisingly, it is chromatin structure, not DNA sequence, that defines a functional centromere in most organisms Human chromosomes that are rearranged to generate two centromeres, and chromosomes into which an extra-large tract of alpha-satellite DNA is introduced, would be expected to have two centromeres but, in fact, often are found to contain just one active centromere This centromere inactivation prevents the chromosome breakage that would occur if two centromeres on the same chromosome were pulled to opposite poles at cell division The formation of neocentromeres can occur in a chromosome that has lost its centromere through a deletion of that segment of DNA An important hallmark of centromeres is the presence of flanking heterochromatin where processes such as gene expression and recombination are often repressed The chromatin structure of centromeres is inherited epigenetically, that is, the sites at which centromeres are formed on newly replicated DNA are not solely determined by the DNA sequence of the parent chromosome The Telomere Telomeres protect the ends of linear chromosomes In some organisms with long telomeres, the G-rich overhang maybe processed into a t-loop. This loop is formed by base-pairing between the single-stranded a overhang and the doublestranded region of the telomere DNA The telomere repeats are binding sites for specific proteins that mark the chromosome ends as natural ends and thereby distinguish them from DNA breaks. This prevents the inappropriate activation of the cell's DNA damage response, which often leads to cell death Telomerase solves the end-replication problem To solve the problem of not being able to complete telomere replication to the very end, most organisms use the enzyme telomerase to add telomeric sequences de novo onto telomeres to extend them The RNA contains a short region that can act as a template to specify the sequence of the telomere repeats that are added onto the chromosome ends. The protein component synthesizes the telomeric DNA ' using the RNA component as a template Telomere-binding proteins are essential for telomere function There are three classes of telomere-binding proteins. End-binding proteins bind to and protect the G-rich overhang at the extreme terminus; double-stranded DNA-binding proteins bind along the length of the telomere sequence The Separation of Chromatin Domains by Boundary Elements Special DNA sites establish the transition between euchromatin and heterochromatin Cells appear to have specific boundaries between euchromatin and heterochromatin, implying that there is a mechanism for maintaining the separation between these two distinct forms of chromatin and preventing one from encroaching the other This rearrangement, known as a chromosomal inversion, also likely removes a barrier element that normally separates the euchromatic and heterochromatic regions, thus allowing heterochromatin to spread into sequences that are usually euchromatic