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The centromere is the part of a chromosome that links sister chromatids. During mitosis, spindle fibers attach to the centromere via the kinetochore. Centromeres were first defined as genetic loci that direct the behavior of chromosomes.

Their physical role is to act as the site of assembly of the kinetochore - a highly complex multiprotein structure that is responsible for the actual events of chromosome segregation - i.e. binding microtubules and signalling to the cell cycle machinery when all chromosomes have adopted correct attachments to the spindle, so that it is safe for cell division to proceed to completion and for cells to enter anaphase.

There are broadly speaking two types of centromeres. "Point centromeres" bind to specific proteins that recognise particular DNA sequences with high efficiency. Any piece of DNA with the point centromere DNA sequence on it will typically form a centromere if present in the appropriate species. The best characterised point centromeres are those of the budding yeast, Saccharomyces cerevisiae. "Regional centromeres" is the term coined to describe most centromeres, which typically form on regions of preferred DNA sequence, but which can form on other DNA sequences as well. The signal for formation of a regional centromere appears to be epigenetic. Most organisms, ranging from the fission yeast Schizosaccharomyces pombe to humans, have regional centromeres.

Regarding mitotic chromosome structure, centromeres represent a constricted region of the chromosome (often referred to as the primary constriction) where two identical sister chromatids are most closely in contact. When cells enter mitosis, the sister chromatids (which represent the two copies of each chromosomal DNA molecule resulting from DNA replication earlier in the cell cycle and packaged by histones and other proteins into chromatin) are linked all along their length by the action of the cohesin complex. It is now believed that this complex is mostly released from chromosome arms during prophase, so that by the time the chromosomes line up at the mid-plane of the mitotic spindle (also known as the metaphase plate), the last place where they are linked with one another is in the chromatin in and around the centromere.

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