Functional Dissection of Human and Mouse POT1 Proteins

Abstract

The single-stranded telomeric DNA binding protein POT1 protects mammalian chromosome ends from the ATR-dependent DNA damage response, regulates telomerase-mediated telomere extension, and limits 5-end resection at telomere termini. Whereas most mammals have a single POT1 gene, mice have two POT1 proteins that are functionally distinct. POT1a represses the DNA damage response, and POT1b controls 5-end resection. In contrast, as we report here, POT1a and POT1b do not differ in their ability to repress telomere recombination. By swapping domains, we show that the DNA binding domain of POT1a specifies its ability to repress the DNA damage response. However, no differences were detected in the in vitro DNA binding features of POT1a and POT1b. In contrast to the repression of ATR signaling by POT1a, the ability of POT1b to control 5-end resection was found to require two regions in the C terminus, one corresponding to the TPP1 binding domain and a second representing a new domain located between amino acids (aa) 300 and 350. Interestingly, the DNA binding domain of human POT1 can replace that of POT1a to repress ATR signaling, and the POT1b region from aa 300 to 350 required for the regulation of the telomere terminus is functionally conserved in human POT1. Thus, human POT1 combines the features of POT1a and POT1b.Mammalian chromosome ends associate with shelterin, the protein complex dedicated to telomere protection and homeostasis (reviewed in references 5 and 20). The DNA of mammalian telomeres consists of long arrays of doublestranded (ds) TTAGGG repeats ending in a single-stranded (ss) protrusion of the 3Ј end, referred to as the 3Ј overhang. The 3Ј-overhang strand can invade the duplex part of telomeres, forming a lariat structure called the t-loop (8). Three shelterin components interact with telomeric DNA. POT1 recognizes ss TTAGGG repeats and therefore can associate with the telomeric 3Ј overhang as well as with the displaced strand at the base of the t-loop. TRF1 and TRF2 cover the duplex part of telomeric DNA, and they recruit the other shelterin components Rap1, TIN2, and TPP1 to telomeres through protein-protein interactions. POT1 cannot localize to telomeres alone but instead requires heterodimerization with TPP1; this interaction is crucial for the association of POT1 with telomeres, as TPP1 tethers POT1 to TRF1 and TRF2 by a TPP1-TIN2 bridge and improves the DNA binding affinity of POT1 (12,19,26).Shelterin masks telomeres from the cellular machinery that recognizes and repairs DNA lesions. If shelterin function is perturbed, DNA damage response factors accumulate at telomeres, leading to the activation of the ATM and ATR kinase signaling pathways (2, 10, 15, 29). Distinct components of shelterin independently repress ATM and ATR signaling: TRF2 is required to prevent ATM activation at telomeres, while POT1 represses the activation of ATR (16). Dysfunctional telomeres trigger various DNA repair reactions and can be processed like dsDNA breaks through nonhomologous end joining or through homology-direc...

How telomeric protein POT1 avoids RNA to achieve specificity for single-stranded DNA

“…Using a filter-binding assay, we determined the K D of hPOT1-D12 to be 6.3 AE 3.5 nM and that for mPOT1A-D12 to be 0.9 AE 0.3 nM, values that agree well with previous reports (10,28). To quantify the defect in binding for a particular dodecamer, we calculated the ratio of the K D for the complex formed by that dodecamer to the K D for the analogous complex formed by D12 (designated as "fold increase of K D " in Table 1).…”

“…We observed that mouse POT1A, the essential POT1 paralog in mice (7), serves as an excellent source of recombinant mammalian POT1, because it can be obtained readily from overexpression in bacteria (unlike hPOT1, which is isolated after expression in baculovirus-infected insect cells). To examine the effects of ribonucleotide substitution within the telomeric sequence on mammalian POT1 binding, we carried out a parallel binding analysis of hPOT1 and mPOT1A with oligonucleotides that contain two telomeric repeats terminating in TAG-3 0 , which is optimal for mPOT1A and hPOT1 binding (27,28).Like hPOT1 (6), mPOT1A failed to bind an all-RNA telomeric dodecamer R12 under conditions in which it formed a stable complex with D12 ( Fig. 1A; oligonucleotide sequences in Table 1).…”

The Role of Error-Prone Alternative Non-Homologous End-Joining in Genomic Instability in Cancer

“…Critically shortened telomeres are recognized as DSBs and are highly susceptible to be repaired by HR or NHEJ pathways (Palm et al, 2009, Rai et al, 2010. However, unequal exchange of telomeric sequences by HR or misrepair by C-and/or ALT-NHEJ, can lead to loss of cell viability or can result in genomic instability and cancer.…”

RPA and POT1

“…3 Among these shelterin components, TRF1 and TRF2 interact with telomeric dsDNA in a sequence-specific manner, whereas POT1, in a complex with TPP1, binds to protection, 20,41,42 they are neither necessary nor sufficient for the localization of POT1 to telomeres. 8,20,43 Indeed, while a point mutation in the OB fold abolished POT1a binding to telomeric ssDNA in vitro, this mutant efficiently localized to telomeres in vivo.…”

“…In both human and mouse, the abilities of POT1 and POT1a/b to bind TPP1 are critical for their functions in telomere protection. 8,13,[42][43][44] A POT1 mutant lacking the TPP1-binding domain failed to localize to telomeres. 8 Furthermore, a POT1 mutant unable to bind TPP1 failed to prevent RPA accumulation at telomeres, 20 suggesting that the role of POT1 in antagonizing RPA binding is dependent upon its interaction with TPP1.…”