NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 8 AUGUST 2006 673

Stirring the POT1: surprises in telomere protectionCarolyn M Price

The main function of mouse POT1 proteins is to regulate G-overhang length, suppress telomere recombination and prevent a telomeric DNA-damage response. They are not needed to prevent G-overhang loss and have only a minor role in preventing telomere fusions.

than anticipated, with different proteins having different roles.

Mammalian telomeres are protected by a number of specialized telomere proteins. The G-overhang seems to be protected by POT1, as just discussed, whereas the rest of the telomeric tract is protected by a six-protein complex called shelterin4 (Fig. 1). Shelterin includes three DNA-binding proteins: TRF1 and TRF2, which bind directly to the duplex T2AG2 repeats, and POT1. POT1 is anchored into the complex by a C-terminal region that is separate from the DNA-binding domain (Fig. 1). TRF2 is key in telomere protection, as loss of TRF2 leads to a dramatic telomere-fusion phenotype. The fusions arise because the nucleotide excision repair nuclease ERCC1/XPF removes the G- overhangs, allowing ligation of the chromosome ends via NHEJ5,6. Although the exact role of mammalian POT1 has remained unclear, both fission yeast and plant POT1 pro-teins are essential for telomere protection. Deletion of S. pombe POT1 leads to rapid loss of telomeric and subtelomeric sequences and widespread cell death7. Cell that survive do so by circularizing their chromosomes. Likewise, expression of a dominant- negative allele of one of the Arabidopsis thaliana POT1 paralogs, Pot2, leads to extreme telomere shortening, telomere fusions and massive genome instability8.

Although humans have only one POT1 gene, mice have two POT1 paralogs (POT1a and POT1b), which encode proteins that are 72% identical to each other and 71%–75%

identical to the human protein. Hockemeyer et al.5 describe the phenotype of POT1a and POT1b single-gene knockouts and a POT1a/POT1b double knockout, whereas Wu et al.6 focus on a POT1a knockout. The bottom line is that POT1b-null mice are viable, but POT1a is essential during embryogenesis. However, analysis of the POT1a knockout was possible because POT1a-null MEFs are viable. POT1b-null MEFs are also viable, but POT1a/POT1b double-knockout MEFs show a rapid growth arrest. Interestingly, when Hockemeyer et al.5 compared the single-knockout MEFs, they found clear differences in phenotype. For example, loss of POT1a, but not POT1b, caused a telomeric DNA-damage response and some telomere fusions. In contrast, loss of POT1b, but not POT1a, caused elongation of the G-strand overhang. Although POT1 paralogs have previously been found in Arabidopsis, mammalian genomes are usually less plastic than plant genomes. Thus, the rapid evolution of the mouse POT1 proteins is striking. Not only has the mouse genome undergone a gene duplication that is missing in mammals such as humans, cows and dogs, as well as in chickens, but the resulting POT1 genes have evolved rap-idly enough to show separation of function.

Analysis of the various POT1-null MEFs has provided some powerful insights into POT1 function. The emerging picture is that although POT1 proteins are required to prevent the telomere from activating a major DNA-damage response, they are not required to protect the G-strand overhang from deg-radation. Instead, they regulate overhang

The 3′ protrusion of a single-stranded DNA rich in guanine residues at the end of a telo-mere, called the G-strand overhang, is a feature conserved among all eukaryotes. Telomeric G-strand–binding proteins are present in a wide range of organisms, includ-ing vertebrates, plants, fission yeast (POT1 proteins), ciliates (TEBPs) and budding yeast (Cdc13)1. These proteins share a structurally conserved DNA-binding motif that recog-nizes the G-strand overhang. The protection of telomeres-1 (POT1) protein and telomere end–binding proteins (TEBPs) are the most closely related, exhibiting sequence identity in their DNA-binding domains. Initial bio-chemical and structural studies of TEBPs and POT1 proteins, and phenotypic analysis of Schizosaccharomyces pombe POT1-deficient cells, suggested that the main function of POT1 proteins is to bind the G-strand overhang and prevent its degradation. Consequently, loss of mammalian POT1 was expected to result in degradation of the G-overhang and maybe also the double-stranded telomeric DNA, thus allowing extensive telomere fusions by non-homologous end joining (NHEJ). However, depletion of human POT1 by short inter-fering RNAs did not cause the expected high level of telomere fusions, and the telomeres became longer rather than shorter. Although these unexpected phenotypes were intriguing, it was unclear whether they indicated different and more complex functions for mammalian POT1 or merely reflected the limitations of short interfering RNA. This conundrum has now been solved by the publication of two papers describing the phenotype that results from POT1 gene disruptions in mouse embry-onic fibroblasts (MEFs)2,3. These papers reveal previously uncharacterized roles for POT1 in telomere protection and indicate that the pro-cess of protecting a telomere is more complex

The author is in the Department of Molecular Genetics, Biochemistry & Microbiology, University of Cincinnati College of Medicine ML0524, 231 Albert Sabin Way, Cincinnati, Ohio 45267, USA.e-mail: carolyn.price@uc.edu

3′5′

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TRF1 TRF2

RAP1TIN2

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Figure 1 Telomere protection by POT1 and the shelterin complex. POT1 binds the G-overhang via two N-terminal OB folds and interacts with shelterin via a C-terminal interaction with TPP1. Shelterin consists of six proteins: TRF1, TRF2, RAP1, TIN2, TPP1 and POT1.

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674 VOLUME 13 NUMBER 8 AUGUST 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY

length by protecting the C-strand from exces-sive resection. Moreover, the POT1 proteins have a relatively minor role in prevent-ing telomere fusions. Although removal of POT1 does cause an increase in fusions, even in the POT1a/POT1b double knockout this involves <5% of the chromosomes. The low fusion frequency makes sense given that the G-overhangs are either maintained or elon-gated, but this phenotype contrasts with the long trains of fused chromosomes that are observed after TRF2 removal.

Although the two POT1 proteins show dif-ferences in function, there is also functional overlap, and their relationship is clearly com-plex. For example, removal of POT1a and POT1b causes a greater DNA-damage response than removal of POT1a alone. However, no response is observed after removal of POT1b alone. Thus, although POT1a and POT1b both help suppress the DNA-damage response, POT1a seems to be the major player. Another complication is that the Hockemeyer and Wu papers describe some substantial differences in the phenotypes of their POT1a-null MEFs. For example, Wu et al.6 find POT1a loss causes overhang elongation, whereas Hockemeyer et al.5 do not. Hockemeyer et al. report a fascinating endoreduplication phenotype, with continued sister- chromatid cohesion resulting in diplo- and quadruplochromosomes (four and eight sister chromatids). Wu et al. instead report a high frequency of telomeric double-minute chromosomes, telomere sister- chromatid exchanges and small telomeric DNA circles, all findings that point to unbridled telomeric recombination. The mice from the two groups seem to have similar genetic back-grounds, but they were created by different gene- targeting strategies, so perhaps the dif-ferences in phenotype occurred because one of the gene- disruption approaches resulted in expression of a dominant-negative or hypo-morphic allele. The two groups are working to resolve this issue.

Removal of TRF2 and POT1 both result in telomere dysfunction and activation of a hearty telomeric DNA-damage response; however, removal of the two proteins results in quite different outcomes in terms of actual telomere damage. Thus, it seems that the two proteins have quite different roles in telomere protection (Fig. 2). A major func-tion of TRF2 is to protect against NHEJ by preventing G- overhang removal. In contrast, POT1 proteins have a fairly minor role in preventing NHEJ and instead are required to regulate overhang length and protect against C-strand degradation. Although these functions are not what might initially have been predicted from the location and

binding specificity of the two proteins, in hindsight they make sense. Removal of POT1 could have left the G- overhang unprotected; however, cells actually contain a number of proteins that bind G-strand telomeric DNA (such as RPA and hnRNPs. Thus, perhaps the reason for having a specialized telomeric G-strand–binding protein is not to protect the DNA from degradation, but to prevent the overhang from activating a DNA-damage response. Binding of RPA during telomere replication could be particularly problematic, as this would lead to recruitment of the ATR kinase. Likewise, removal of TRF2 might have been expected to allow rapid degradation of the telomeric tract rather than selective loss of the G-overhang. Nevertheless, TRF1 and associated proteins are presumably sufficient to protect the dsDNA, and given that TRF2, but not TRF1, shows some preference for the end of the telomeric duplex, it makes sense for TRF2 to be adapted for repulsing nucle-ases from the overhang.

This leaves us with the difference in phenotype between mouse and fission yeast POT1-null cells. Why does POT1 removal cause complete telomere loss in S. pombe but leave mouse telomeres essentially intact? Perhaps the answer lies in the combined abili-ties of both G-overhang– and double-stranded telomere–binding proteins to resist nuclease attack. The phenotype of the mouse POT1a/POT1b double knockout is actually rather similar to the phenotype of the Saccharomyces cerevisiae Cdc13 temperature-sensitive mutant,

in that both cause C-strand resection and cell-cycle arrest. The main difference is that the extent of C-strand resection is much greater in budding yeast. This suggests that although the G-overhang–binding proteins serve as a first line of defense against 5′→3′ nucleases, the double-stranded telomere–binding pro-teins are an important backup. The mouse telomere is protected by the hefty six-protein shelterin complex, which contains two dedi-cated dsDNA-binding proteins. In contrast, yeast telomere protein complexes contain only a single dsDNA-binding protein and one or two interaction partners. The S. cerevisiae RAP1 complex seems better able to protect the telomere than the S. pombe Taz1 complex: in the absence of G-overhang–binding proteins, S. cerevisiae telomeres are protected from 3′→5′ but not 5′→3′ nucleases, whereas S. pombe telomeres appear to be protected against neither. Thus, the take-home message seems to be that cooperation is the only true line of defense at the end.

1. Wei, C. & Price, M. Cell. Mol. Life Sci. 60, 2283–2294 (2003).

2. Hockemeyer, D., Daniels, J.-P., Takai, H. & De Lange, T. Cell 126, 63–77 (2006).

3. Wu, L. et al. Cell 126, 49–62 (2006).4. de Lange, T. Genes Dev. 19, 2100–2110 (2005).5. Zhu, X.D. et al. Mol. Cell 12, 1489–1498 (2003).6. Celli, G.B. & de Lange, T. Nat. Cell Biol. 7, 712–718

(2005).7. Baumann, P. & Cech, T.R. Science 292, 1171–1175

(2001).8. Shakirov, E.V., Surovtseva, Y.V., Osbun, N. & Shippen,

D.E. Mol. Cell. Biol. 25, 7725–7733 (2005).

Homologousrecombination

T-loop

DNA damageresponse

POT1

POT1

POT1

TRF2

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C-strand resection/G-overhang elongation

Telomere doubleminutes/circles

Overhang removalNHEJ

Figure 2 Different modes of end protection by POT1 and TRF2.

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