Volume 5, Issue 34 January 2008
The Copy Machine of the Cell
Michael Botchan is a professor of biochemistry and molecular biology and a faculty affiliate of QB3. photo credit: Ed Ralston
There comes a time in many a cell's life when it feels the need to reproduce. But before it can split into two, it must fashion a second set of genetic instructions to pass on to the new cell.
When Berkeley professor of biochemistry and molecular biology Mike Botchan first began studying chromosome copying, basic questions about the process remained unknown. He wanted to understand how and where DNA replication began. Over the past three decades, Botchan has been instrumental in piecing together the story of what he calls "the elaborate dance of replication."
Botchan began by studying viruses, the simplest of all life forms. These microbes contain relatively few genes in their chromosome, borrowing much of the machinery needed to duplicate their own DNA from host cells. Because the viral chromosome constitutes a tiny fraction of the DNA in a host cell, its chances of encountering the necessary copying proteins are low. To compensate, Botchan found, viruses use a DNA sequence that binds strongly to these replication proteins.
To decipher the string of events required to start replication, Botchan mapped the initiation site-a place on a chromosome where replication begins-in a virus. He found that a certain DNA sequence attracts a virus protein involved in replication initiation. Only then can the virus helicase, which unwinds and separates the strands of DNA, bind to the chromosome and start unraveling DNA. The unwinding process attracts cellular proteins needed to copy the virus chromosome.
Botchan found a protein complex that higher animals use to locate where DNA replication should begin. In a fruit fly egg, this complex (red) can be seen where there is both DNA (blue) and new strands of DNA are being replicated from component nucleotides (green). Image credit: courtesy Michael Botchan
But do more complex organisms, such as insects and humans, copy their DNA in a similar fashion? To find out, Botchan studied a case of unchecked DNA replication in fruit fly embryos. The cells that go on to form the fly's eggshell duplicate certain sections of their DNA with astonishing rapidity, initiating replication at many sites at once. In these cells, Botchan found and characterized a complex of proteins that finds the initiation site and prepares the chromosome so that a core replication machine can be assembled there. The core replication machine includes a six-protein complex used at all DNA replication sites. Several of these proteins form a pinwheel structure that encircles DNA, while another links to the polymerase enzyme that "reads" the sequence. In cells actively copying their DNA, all of these proteins are located right on top of one another.
Botchan found that this core DNA replication protein complex has no preference for particular stretches of DNA. This explains why DNA duplication can occur at many sites simultaneously in fly embryos. Early on in development, a fly's chromosomes resemble long necklaces of DNA. But as the embryo matures, its chromosomes curl into spirals resembling a coiled telephone cord. Now replication can begin only at well-defined initiation sites. This is because the core replication complex can only bind to open regions of the chromosome where other cell-specific protein complexes help it to attach.
A chromosome's three-dimensional coils also help determine which genes are turned on and shut off. "The process of getting the chromosome prepared for gene expression and replication go hand in glove," Botchan says. "The same factors that recognize certain DNA sequences for initiation also make the region of the chromosome available for protein transcription."
Botchan's work, along with research by Berkeley biologists Eva Nogales and James Berger, helps prove that DNA replication has changed very little across evolution. "All three kingdoms of life share a basic core machinery that assembles on DNA and prepares it for unwinding," Botchan says. Organisms ranging from E. coli to fruit flies, they find, have nearly identical chromosome copying methods, cementing the relationship of all life forms back to that first ancestral cell.