De Lange, who is head of the Laboratory of Cell Biology and Genetics, is now pushing to understand how this duplicitous enzyme, known as ERCC1/ XPF, is itself managed. The answer, she says, might unlock the mystery of how a cell controls its chromosomes — a process crucial to every organism’s development and day-to-day function.

While the chromosomes of bacteria are circular in shape, the chromosomes of humans and other more complex organisms are linear and are sealed at their ends by specialized protein-DNA complexes known as telomeres. In addition to protecting the ends of chromosomes — think of the plastic at the end of a shoelace that keeps it from fraying — telomeres act as a sort of molecular clock, ticking down the number of times a cell can replicate. With every cell division, telomeres shorten in length, and de Lange has shown that a cell ceases to reproduce itself when its telomeres become too short to protect the ends of chromosomes.

Four years ago, de Lange and her group, in collaboration with researchers at the University of North Carolina at Chapel Hill, found that telomeres form closed loops, called t-loops, at their ends. The loops act as protective caps on the end of telomeres, which in turn protect the end of chromosomes. A protein complex called TRF2 protects the telomeres, probably by stimulating formation of these protective loops. Without the loops, cells mistake the exposed chromosome ends for sites of DNA damage; when they attempt to repair them, the cells die. (A related protein complex, TRF1, regulates telomere length.)

Normally hidden within a t-loop is a single strand of DNA that acts like a latch to secure the loop structure. But de Lange and her team noticed that when telomeres lose TRF2, this latch quickly disappears. “We were keenly interested in trying to find out what is responsible for making this strand disappear,” she says, “and thought it might be a nuclease, which can quickly degrade DNA.” De Lange has already identified several interacting partner proteins within the TRF1 and TRF2 complexes that play very specific roles in telomere regulation.

The group tested several recognized DNA nuclease enzymes in a series of experiments, led by first author Xu-Dong Zhu, then a postdoctoral researcher in de Lange’s lab and now assistant professor at McMaster University. The scientists found, to their surprise, that the enzyme responsible for destroying the overhang strand was ERCC1/XPF, a well-known repair tool that cleaves away portions of DNA strands that have been damaged by ultraviolet light. The rare individual born with defective ERCC1/XPF suffers from a skin disease called xeroderma pigmentosa, often accompanied by persistent skin cancer.

“We never expected ERCC1/XPF to be the culprit nuclease because it had only been studied in the context of UV damage. These proteins had never been implicated at telomeres in human cells or cells from any other organism,” says de Lange who is Rockefeller’s Leon Hess Professor. “Yet we found that when TRF2 is inhibited and telomeres lose their protection, ERCC1/ XPF is one of the factors involved in repairing what the cell thinks is a site of damage. It removes the overhang and then the ends of the chromosomes are fused together.”

De Lange and Zhu turned to research-ers in The Netherlands and Denmark for ERCC1/XPF deficient mouse cells with which to run further tests. For de Lange, this alliance was a scientific homecoming of sorts: not only was she born and raised in The Netherlands, but her collaboration was with Jan H.J. Hoeijmakers of Erasmus Medical Center, who identified in 1980 the genes that first drew her to the study of telomeres. “The first experiment I did with those genes showed us they had located at telomeres, and that is how my interest in telomeres began,” de Lange recalls.

Using the deficient mouse cells, the researchers found that the overhang remained intact even without TRF2, pinpointing ERCC1/XPF as the protein responsible.

But further analysis by Zhu found ERCC1/XPF was not only associated with telomeres in the cell, but also within the TRF2 complex. “This was a big puzzle. Here is a nuclease that can threaten telomeres when things are off, when TRF2 is inhibited, and yet TRF2 brings that protein to the telomeres,” says de Lange. “So this nuclease must also have a protective role to play otherwise it wouldn’t be located in the telomere protein complex.”

Zhu then noticed that when ERCC1/ XPF-deficient cells divided, they produced loose pieces of chromosomal material with telomeric DNA on them. De Lange speculates that these small, so-called double minute chromosomes were created when the repeat sequence of DNA found in telomeres recombined with similar sequences elsewhere on the chromosome. The role of ERCC1/XPF must therefore be to prevent these recombination events by cleaving away these mistakes. That was the protective effect: cells with the ERCC1/XPF did not produce the mutant chromosome bodies.

So, unexpectedly, ERCC1/XPF plays two very different roles at chromosome ends. Under normal circumstances it prevents errant recombinations during chromosome division. But when the telomere cap is no longer in place, ERCC1/XPF likely has a very negative effect, seeming to destroy the telomere by consuming its latch.

“It’s a delicate balancing act,” says de Lange. “The telomere seems to both protect itself from DNA repair factors but also, at the same time, use those DNA repair factors for its own purposes.

“Now we have to sort out what controls this nuclease, to learn how it knows when to perform which of its functions. The whole key to understanding telomeres is in understanding how this regulation occurs.”

January 30, 2004