Mechanisms of Telomere Protection and Deprotection in Human Cells

Abstract

Telomeres, the nucleo-protein complexes at the ends of linear chromosomes, have critical roles in genome stability, cancer, and aging. Early work by B. McClintock and H.J. Muller demonstrated that eukaryotic chromosome ends contain specialized structures that prevent recognition and processing by the DNA repair machinery. The importance of these structures is illustrated by studies showing that loss of chromosome end protection results in massive genome instability and cell death. Although Muller and McClintock's initial observations were made several decades ago, little progress has been made in understanding the molecular markers that distinguish naturally occurring chromosome ends from de novo DNA double strand breaks, especially in humans. Using a novel system to specifically target proteins of interest to human telomeres, we have uncovered a role for hRAP1 in protecting telomeres from non-homologous end joining (NHEJ). We find that telomeric DNA containing hRAP1, but not TRF2, is protected from NHEJ in vitro. Furthermore, we show that telomeres containing TRF2 but not hRAP1 can be fused by NHEJ in vivo, and we also demonstrate that targeting hRAP1 to telomeres in vivo, even when TRF2 is not detected, is sufficient to protect telomeres from NHEJ. These results identify hRAP1 as a critical mediator of telomere protection and genome stability in humans. Related to this work, we have also identified a new type of telomere dysfunction associated with semi-conservative replication stress at human telomeres. This new type of telomere dysfunction is telomerase and NHEJ-independent and may require the RecQ helicase WRN for its formation, suggesting that it is related to telomere entanglements observed upon induction of replication stress in fission yeast. The finding that this type of dysfunction is conserved from yeast to man is a testament to the underappreciated role of semi-conservative DNA synthesis in maintaining telomere structure and function.