Frederick Alt, PhD

Frederick Alt, PhD, co-chief of the Division of Molecular Medicine at Children's Hospital Boston and a Howard Hughes Medical Institute investigator, and his colleagues published their discovery in the August 22, 2007, advance online publication of the journal Nature. First author was Catherine Yan, PhD, in the Alt laboratory at Children's and Harvard Medical School. The finding highlights the surprising importance of the alternate DNA repair mechanism in one of the most fundamental processes in the immune system, said the researchers. Also, they said, there are hints that the repair mechanism might play a role in the abnormal chromosomal rearrangements that underlie lymphomas.

The researchers explored how broken DNA is repaired in B cells, which are specialized immune cells that produce antibodies. Antibodies are proteins that recognize and attach to telltale components, called antigens, of foreign invaders such as viruses and bacteria, in order to destroy them. To generate specialized antibodies that can transport their antigen cargoes to specific destinations, B cells use a process called class switch recombination (CSR), in which genes for the antibodies are snipped and rearranged to produce different antibody types. The snipped-apart DNA must then be rejoined. Researchers have believed that B cells use a process called classical non-homologous end-joining for such repair.

To test whether this classical process is, indeed, involved in CSR, the researchers genetically knocked out two critical components of the end-joining machinery, proteins known as XRCC4 and Ligase 4.

Eliminating these proteins reduced the B cells' ability to rejoin DNA broken in CSR, suggesting that classical end-joining was important. But lots of class switching still occurred, and the researchers speculated that an alternate method of rejoining DNA ends must exist.

When they analyzed what kind of DNA breaks were repaired in the absence of CSR, they found that the alternate end-joining mechanism appeared to work only on pairs of broken DNA ends that had microhomologies. Microhomologies are short regions of overhanging single-stranded DNA that give the two broken DNA segments a complementarity -- like two pieces of a puzzle fitting together.

The researchers also plan studies to identify the components of the alternate end-joining machinery, said Alt. "While there could be some novel components, we think this pathway probably uses a subset of factors that are used for other DNA repair pathways," he said. "Once we have identified those components, we can dissect in normal cells what specialized role this repair pathway plays," he said.

Importantly, the Alt lab has generated evidence that the alternate pathway might have detrimental effects--catalyzing aberrant chromosomal rearrangements, or translocations, that underlie lymphomas that arise in mice lacking components of the classical pathway and the p53 checkpoint protein.

"We don't really know if this is a 'bad' pathway," said Alt. "However, our studies indicate that in the absence of the classical joining process, up to twenty percent of the breaks in CSR that do not get repaired get joined to other chromosomes as translocations," said Alt. "So, for some reason the alternate pathway very effectively catalyzes translocations." He also noted that studies done by others have indicated that the translocations that underlie many B cell lymphomas show microhomologies, which implies that they are being produced by the alternate pathway.

The alternative pathway had already been implicated from other studies but was not thought to function on chromosomal DNA. "Based on our current findings, this pathway will clearly be given more serious consideration, both for its ability to repair normal chromosomal DNA damage and for its ability to catalyze unwanted end-joins such as translocations," said Alt.

Contact:
Anna Gonski
617-355-6420
anna.gonski@childrens.harvard.edu

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