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Sequence-specific processing of telomeric 3' overhangs by the Werner syndrome protein exonuclease activity.

Li B, Reddy S, Comai L - Aging (Albany NY) (2009)

Bottom Line: The findings that cells lacking WRN activity display accelerated telomere shortening and WRN can be detected at chromosome ends suggest that this protein participates in some aspects of telomere metabolism.This activity is strictly dependent on the presence of telomeric sequences in both the duplex DNA and 3' overhang DNA segment and is strongly inhibited by the telomeric factor POT1 but not TRF2.These data demonstrate that WRN processes telomeric DNA substrates with a 3' single-stranded overhang with high specificity and suggest that this protein could influence the configuration of telomere ends prior to the formation of a protective t-loop structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Institute for Genetic Medicine, University of Southern California, Los Angeles, CA 90033, USA.

ABSTRACT
Werner syndrome is a premature aging disease caused by loss of function mutations in the Werner syndrome protein (WRN) gene. WRN is a RecQ helicase that in contrast to every other member of this family of proteins possesses an exonuclease activity. The findings that cells lacking WRN activity display accelerated telomere shortening and WRN can be detected at chromosome ends suggest that this protein participates in some aspects of telomere metabolism. In this study we examined the impact of WRN on telomeric substrates with a 3' single-stranded overhang in vitro and show that WRN has sequence-specific exonuclease activity that removes several nucleotides inward with a periodical pattern from the 3' end of the telomeric overhang. This activity is strictly dependent on the presence of telomeric sequences in both the duplex DNA and 3' overhang DNA segment and is strongly inhibited by the telomeric factor POT1 but not TRF2. These data demonstrate that WRN processes telomeric DNA substrates with a 3' single-stranded overhang with high specificity and suggest that this protein could influence the configuration of telomere ends prior to the formation of a protective t-loop structure.

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WRN exonuclease resects the 3' single-stranded overhang of telomeric DNA substrates. (A) 100 to 400 fmol of purified                                            recombinant wild-type WRN or exonuclease mutant WRN (WRN D82A) were                                            incubated with 5'-32P-labeled, 15 nt 3'-overhang DNA substrates                                            containing non-telomeric sequences (lanes 1-5) or telomeric (TTAGGG)                                            repeats (lanes 6-10) at 37°C for 10 min. The reaction products were                                            analyzed by 12% polyacrylamide-urea denaturing gel and autoradiography (lane                                                    1 to 4, 100, 200, 300, and 400 fmol of WRN; lane 5,                                            non-telomeric DNA substrate; lane 6 to 9, 100, 200, 300, and 400                                            fmol of WRN; lane 10, telomeric DNA substrate; lane 11, (TTAGGG)                                            repeats molecular size markers, lane 12 to 15, 100, 200, 300, and                                            400 fmol of exonuclease mutant WRN(D82A).                                            (B) 100 to 400 fmol of purified recombinant WRN were incubated with                                            5'-32P-labeled, non-telomeric (lanes 1-5) or telomeric (lanes                                                    6-10) DNA substrates with 27 nt 3'-overhang at 37°C for 10 min. The                                            reaction products were analyzed by 12% polyacrylamide-urea denaturing gel                                            and autoradiography (lane 1 to 4; 100, 200, 300, and 400 fmol of                                            WRN; lane 5, non-telomeric DNA substrate; lane 6 to 9, 100,                                            200, 300, and 400 fmol of WRN; lane 10, telomeric DNA substrate,                                                    lane 11, (TTAGGG) repeats molecular size markers.
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Figure 1: WRN exonuclease resects the 3' single-stranded overhang of telomeric DNA substrates. (A) 100 to 400 fmol of purified recombinant wild-type WRN or exonuclease mutant WRN (WRN D82A) were incubated with 5'-32P-labeled, 15 nt 3'-overhang DNA substrates containing non-telomeric sequences (lanes 1-5) or telomeric (TTAGGG) repeats (lanes 6-10) at 37°C for 10 min. The reaction products were analyzed by 12% polyacrylamide-urea denaturing gel and autoradiography (lane 1 to 4, 100, 200, 300, and 400 fmol of WRN; lane 5, non-telomeric DNA substrate; lane 6 to 9, 100, 200, 300, and 400 fmol of WRN; lane 10, telomeric DNA substrate; lane 11, (TTAGGG) repeats molecular size markers, lane 12 to 15, 100, 200, 300, and 400 fmol of exonuclease mutant WRN(D82A). (B) 100 to 400 fmol of purified recombinant WRN were incubated with 5'-32P-labeled, non-telomeric (lanes 1-5) or telomeric (lanes 6-10) DNA substrates with 27 nt 3'-overhang at 37°C for 10 min. The reaction products were analyzed by 12% polyacrylamide-urea denaturing gel and autoradiography (lane 1 to 4; 100, 200, 300, and 400 fmol of WRN; lane 5, non-telomeric DNA substrate; lane 6 to 9, 100, 200, 300, and 400 fmol of WRN; lane 10, telomeric DNA substrate, lane 11, (TTAGGG) repeats molecular size markers.

Mentions: In previous studies, we and others have demonstrated that standard double-stranded DNA substrates with a 3' overhang are not significantly processed by WRN exonuclease in vitro [12,13,38,39]. Yet, the presence of WRN at telomeres prompted us to test whether telomeric sequences with a 3' single-stranded overhang are particularly susceptible to WRN-mediated processing. For this purpose, non-telomeric or telomeric DNA substrates with a 15 nucleotides 3' overhang were incubated with increasing amounts of purified WRN in cell-free exonuclease assays. DNA products from these reactions were separated by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. In agreement with our prior data [38], a non-telomeric double-stranded DNA substrate with a 3' overhang is not degraded by WRN (Figure 1). In contrast, we observed that WRN exonuclease removes several nucleotides from most of the 3' overhangs of the telomeric substrate. Notably, 3' processing of the telomeric substrate is not highly processive, as exonuclease activity slows down as it moves across the GGG trinucleotide repeats and in proximity of the junction between single-stranded and double-stranded DNA, and produces weaker degradation further inward (Figure 1 and Supplementary Figure 1). Thus, the majority of the processing occurs within the single-stranded region of the DNA substrate leading to the generation of products with a shorter 3' overhang. This striking profile of DNA proces- sing is strictly dependent on a functional WRN, since degradation of the 3' overhang from the telomeric substrate is abolished when DNA is incubated with a mutant WRN lacking exonuclease activity (WRN-D82A).


Sequence-specific processing of telomeric 3' overhangs by the Werner syndrome protein exonuclease activity.

Li B, Reddy S, Comai L - Aging (Albany NY) (2009)

WRN exonuclease resects the 3' single-stranded overhang of telomeric DNA substrates. (A) 100 to 400 fmol of purified                                            recombinant wild-type WRN or exonuclease mutant WRN (WRN D82A) were                                            incubated with 5'-32P-labeled, 15 nt 3'-overhang DNA substrates                                            containing non-telomeric sequences (lanes 1-5) or telomeric (TTAGGG)                                            repeats (lanes 6-10) at 37°C for 10 min. The reaction products were                                            analyzed by 12% polyacrylamide-urea denaturing gel and autoradiography (lane                                                    1 to 4, 100, 200, 300, and 400 fmol of WRN; lane 5,                                            non-telomeric DNA substrate; lane 6 to 9, 100, 200, 300, and 400                                            fmol of WRN; lane 10, telomeric DNA substrate; lane 11, (TTAGGG)                                            repeats molecular size markers, lane 12 to 15, 100, 200, 300, and                                            400 fmol of exonuclease mutant WRN(D82A).                                            (B) 100 to 400 fmol of purified recombinant WRN were incubated with                                            5'-32P-labeled, non-telomeric (lanes 1-5) or telomeric (lanes                                                    6-10) DNA substrates with 27 nt 3'-overhang at 37°C for 10 min. The                                            reaction products were analyzed by 12% polyacrylamide-urea denaturing gel                                            and autoradiography (lane 1 to 4; 100, 200, 300, and 400 fmol of                                            WRN; lane 5, non-telomeric DNA substrate; lane 6 to 9, 100,                                            200, 300, and 400 fmol of WRN; lane 10, telomeric DNA substrate,                                                    lane 11, (TTAGGG) repeats molecular size markers.
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Figure 1: WRN exonuclease resects the 3' single-stranded overhang of telomeric DNA substrates. (A) 100 to 400 fmol of purified recombinant wild-type WRN or exonuclease mutant WRN (WRN D82A) were incubated with 5'-32P-labeled, 15 nt 3'-overhang DNA substrates containing non-telomeric sequences (lanes 1-5) or telomeric (TTAGGG) repeats (lanes 6-10) at 37°C for 10 min. The reaction products were analyzed by 12% polyacrylamide-urea denaturing gel and autoradiography (lane 1 to 4, 100, 200, 300, and 400 fmol of WRN; lane 5, non-telomeric DNA substrate; lane 6 to 9, 100, 200, 300, and 400 fmol of WRN; lane 10, telomeric DNA substrate; lane 11, (TTAGGG) repeats molecular size markers, lane 12 to 15, 100, 200, 300, and 400 fmol of exonuclease mutant WRN(D82A). (B) 100 to 400 fmol of purified recombinant WRN were incubated with 5'-32P-labeled, non-telomeric (lanes 1-5) or telomeric (lanes 6-10) DNA substrates with 27 nt 3'-overhang at 37°C for 10 min. The reaction products were analyzed by 12% polyacrylamide-urea denaturing gel and autoradiography (lane 1 to 4; 100, 200, 300, and 400 fmol of WRN; lane 5, non-telomeric DNA substrate; lane 6 to 9, 100, 200, 300, and 400 fmol of WRN; lane 10, telomeric DNA substrate, lane 11, (TTAGGG) repeats molecular size markers.
Mentions: In previous studies, we and others have demonstrated that standard double-stranded DNA substrates with a 3' overhang are not significantly processed by WRN exonuclease in vitro [12,13,38,39]. Yet, the presence of WRN at telomeres prompted us to test whether telomeric sequences with a 3' single-stranded overhang are particularly susceptible to WRN-mediated processing. For this purpose, non-telomeric or telomeric DNA substrates with a 15 nucleotides 3' overhang were incubated with increasing amounts of purified WRN in cell-free exonuclease assays. DNA products from these reactions were separated by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. In agreement with our prior data [38], a non-telomeric double-stranded DNA substrate with a 3' overhang is not degraded by WRN (Figure 1). In contrast, we observed that WRN exonuclease removes several nucleotides from most of the 3' overhangs of the telomeric substrate. Notably, 3' processing of the telomeric substrate is not highly processive, as exonuclease activity slows down as it moves across the GGG trinucleotide repeats and in proximity of the junction between single-stranded and double-stranded DNA, and produces weaker degradation further inward (Figure 1 and Supplementary Figure 1). Thus, the majority of the processing occurs within the single-stranded region of the DNA substrate leading to the generation of products with a shorter 3' overhang. This striking profile of DNA proces- sing is strictly dependent on a functional WRN, since degradation of the 3' overhang from the telomeric substrate is abolished when DNA is incubated with a mutant WRN lacking exonuclease activity (WRN-D82A).

Bottom Line: The findings that cells lacking WRN activity display accelerated telomere shortening and WRN can be detected at chromosome ends suggest that this protein participates in some aspects of telomere metabolism.This activity is strictly dependent on the presence of telomeric sequences in both the duplex DNA and 3' overhang DNA segment and is strongly inhibited by the telomeric factor POT1 but not TRF2.These data demonstrate that WRN processes telomeric DNA substrates with a 3' single-stranded overhang with high specificity and suggest that this protein could influence the configuration of telomere ends prior to the formation of a protective t-loop structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Institute for Genetic Medicine, University of Southern California, Los Angeles, CA 90033, USA.

ABSTRACT
Werner syndrome is a premature aging disease caused by loss of function mutations in the Werner syndrome protein (WRN) gene. WRN is a RecQ helicase that in contrast to every other member of this family of proteins possesses an exonuclease activity. The findings that cells lacking WRN activity display accelerated telomere shortening and WRN can be detected at chromosome ends suggest that this protein participates in some aspects of telomere metabolism. In this study we examined the impact of WRN on telomeric substrates with a 3' single-stranded overhang in vitro and show that WRN has sequence-specific exonuclease activity that removes several nucleotides inward with a periodical pattern from the 3' end of the telomeric overhang. This activity is strictly dependent on the presence of telomeric sequences in both the duplex DNA and 3' overhang DNA segment and is strongly inhibited by the telomeric factor POT1 but not TRF2. These data demonstrate that WRN processes telomeric DNA substrates with a 3' single-stranded overhang with high specificity and suggest that this protein could influence the configuration of telomere ends prior to the formation of a protective t-loop structure.

Show MeSH
Related in: MedlinePlus