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Lecture 12, part 1 of 2
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October 9, 2018


Lodish et al., Molecular Cell Biology, Chapter 9.6
Murray & Szostak (1983) Nature 305:89.

A.How DNA replication works
.Origins of Replication (Autonomously Replicating Sequences)

1. Circularity of prokaryotic chromosomes simplifies the process of DNA replication.
2. Without special provisions for replicating the ends, linear chromosomes would lose some sequence from the ends every cell cycle. 

While our task of building a chromosome has been of enormous scope, the main thing we've done so far is simply to describe how DNA is packaged in chromosomes, and some of the functional ramifications of that packaging. We still need to identify some important functional features of chromosomes that make it possible for them to  replicate and  segregate. We will describe a minimal set of features required for normal replication and segregation. These are   centromeres, origins of replication, and telomeres.

A.How DNA replication works

Read before class: How DNA replication works

B.Origins of Replication (Autonomously Replicating Sequences)

Origins of replication are sites at which DNA replication is initiated. Typically, replication origins contain internal repeats, and are rich in A-T base pairs. Since A-T base-pairing is weaker than G-C base-pairing, A-T rich helices make it easier for helicases to open the helix, allowing primases and other enzymes access to each strand.

Eukaryotic chromosomes contain numerous replication origins, allowing for DNA synthesis to proceed in parallel at many sites simultaneously.

The electron micrograph at right shows several replicons, each originating from a different origin (arrows).

Each replicon has two replication forks, moving in opposite directions. Ultimately, replication forks meet, until replication of each template strand is complete.

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Cloning of replication origins

Functionally, replication origins are defined by their ability to confer stable inheritance of a selectable marker from one cell generation to the next.

A series of experiments from Jack Szostak's lab helped to define the critical components of chromosomes. These experiments all had the same steps:

1. Transform yeast, deficient in Leucine production (Leu-)  with an artificial construct
2. Isolate a colony on complete media (permissive conditions)
3. Grow in complete media for several generations
4. Transfer to minimal media without Leucine
5. Identify clones that grow without Leucine

If you transfect into yeast a plasmid containing a selectable marker, in this case a LEU gene (Leucine biosynthesis) into Leu- yeast, the cells will not grow, even though you have given them the correct gene. But if you randomly clone yeast sequences into this same plasmid and select on minimal media, you can recover a few clones that are able to grow (ie. synthesize leucine). The inserts contained in these surviving plasmids are origins of replication, referred to in yeast as autonomously replicating sequences (ARS).
ARS have been identified in a variety of other species (eg. humans, Drosophila, maize, tobacco, even bacteria) by virtue of their ability to replicate in yeast.


B. Centromeres

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Basically the same strategy has been used to clone centromeric sequences. A plasmid containing a selectable marker and an ARS sequence will replicate in culture, but will be lost from some lines of cells in the absence of selection. Yeast genomic DNA is randomly cloned into the plasmid containing LEU and ARS, clones can be identified that stably maintain the plasmid, even in the absence of selection. These plasmids contain centromere sequences (CEN). Segregation of CEN plasmids occurs in a Mendellian fashion. That is, 100% of the progeny are Leu+ if they contain CEN.
The attachment of a chromosome to the mitotic spindle fibers occurs at the centromeric sequence.
It is probably not a coincidence that the length of the yeast  CEN sequence necessary for binding centromere attachment is 220bp, which spans about 20nm, which is the width of a spindle fiber. DNAse sensitivity studies have indicated that this region is  free of nucleosomes, presumably to enable the spindle attachment to occur. CEN sequences are complexed with the proteins of the kinetochore complex, which facilitate attachment to the spindle fibers.
Centromeres tend to be surrounded by highly-repetitive DNAs, referred to as   satellite DNAs. Satellite DNAs may confer different coiling properties to the centromeric regions of chromosomes.

Detail of a positive staining centromere region of a C-banded human chromosome. (C banding preferrentially stains constitutive heterochromatin.) The fibrous organization of this region is still apparent but is either covered by, or embedded in, an amorphous matrix. (Magnification x 64,000.)

Chromosomes and Chromatin, Vol. II (1988) Ed. K.W.Adolph. CRC Press. Fig. 14 pg. 67

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last  page PLNT3140 Introductory Cytogenetics
Lecture 12, part 1 of 2
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