Molecular Genetics

Though centromeres differ widely in structure, they all serve as sites for organizing kinetochores.

Facts

• Replicated chromosomes, having duplicated centromeres, have duplicated kinetochores. In mitosis and meiosis II, these kinetochores must be oppositely oriented with respect to the long axis of the chromosome for proper segregation to occur.
• Three types of kinetochores can be recognized. Any one species of organism has but one type.
  • In budding yeast, the kinetochores are so small as not to be seen microscopically. They attach a single microtubule.
  • In metazoans, the kinetochores are 100-500 nm in diameter and attach to from 3 to 40 microtubules.
  • In some organisms, such as some plants and Caenorhabditis elegans, kinetochores are distributed over the entire surface of chromosomes. This organization is called holocentric (as opposed to acro-, meta- and telocentric forms).

	Centromeres of mammalian chromosomes have 3 morphological domains: kinetochore, central, and pairing.
The kinetochore consists of inner and outer electron dense plates, separated by a translucent middle zone. The outer plate is adjacent to a fibrous corona containing tubulin. The central zone contains repetitive DNA elements.

• CREST antibodies against some centromere-associated proteins are obtained from patients with certain autoimmune conditions. They have been used to isolate CEN-specific proteins and to locate them in the cell.
• CENP-B, an acidic serine-rich protein, is found in the central zone. CENP-B binds alpha satellite DNA. The binding sequence is CTTCGTTGGAAACGGGA and is present in a subset of alphoid repeats. HMG-1 also binds to alphoid satelite repeats.
• In humans, CENP-A , a histone H3-like protein, and CENP-C are located in a ring around the kinetochore.
  • A homologue of CENP-A exists in all eukaryotes studied. The general name for the homologues is CENH3.
  • In human cytologically stable dicentric chromosomes, only one of the two centromeres has CENP-A, as detected by immunofluorescence. It is the functional centromere (ref)
  • Mutations in the gene for CENH3 in yeast, worms, flies and mammals lead to disruption of mitosis. In these cases, other usually centromeric proteins are located in places other than the centromere (ref) .
  • CENP-A and H3 nucleosomes alternate along the chromatin fiber, but only CENP-A is exposed to the surface.
• INCENP proteins are located in the pairing domain, but are released from the chromosomes at the onset of anaphase.
• Microinjection of anti CENP-B causes arrest of cells between G2 and M. Microinjection of anti CENP-E causes metaphase arrest.
• Use of RNA interference to deplete the levels of condensin II (the only condensin around centromeres in humans) results in a shift of kinetochores from the desired opposite orientation and missegregation.
• Kinetochores are located in the primary constrictions of chromosomes.
  • .As a result, they are in a depression which constricts the angle at which microtubules can bind to the kinetochore.
  • The constriction can be ablated by mutants in genes for Plk1 or Aurora-B. These mutations also result in non-disjunction. These proteins are protein kinases. Their targets are currently uncertain.
• During meiosis I, sister kinetochores need to be oriented to the same pole. Genetic experiments suggest that Rec8 must be present during replication to achieve this monopolar orientation, but it is not sufficient. Other factors implicated as monopolins in budding yeast are Mam1, Csm1, and Lrs4 (rev).

Interpretations

• Attachment of chromosomes to spindles is mediated by a DNA-multiprotein complex.
• The CENH3 protein is fundamental to the kinetochore interaction with DNA.
• A coherent picture of the interactions requires more information.
• Kinetochore orientation is critical for proper segregation (rev).

Further information

• Anaphase released INCENP proteins may be involved in cell division since they form a ring at the anaphase plate.
• See a diagrammatic representation of the microtubule-kinetochore-centromere structure.
• Attachment of a pair of kinetochores to microtubules can be amphitelic (to opposite poles), syntelic (to the same pole), monotelic (only one kinetochore connected) or mesotelic (one kinetochore connected to both poles (rev).
• Genetic searches for mutations affecting kinetochores will reveal more about their structure and function.
• In Saccharomyces cerevisiae, the structure of the centromere appears to be (ref) that of a modified nucleosome.
  • The yeast CENP-A homolog, Cse4p, also resembles the H3 histone.
  • Like H3, it interacts with H4.
  • Cse4p nucleosomes are found only at the centromere.
  • Cse4p interacts sequence specifically with the CDEII element.
  • Wrapping CDEII around nucleosomes brings CDEI and CDEII elements together, a result expected from genetic observations.

This is page 1365 of Molecular Genetics by Ulrich Melcher, © 1997, 1998, 1999, 2004