Telomere repeat DNA forms a large non-covalent complex with unique cohesive properties which is dissociated by Werner syndrome DNA helicase in the presence of replication protein A.
We describe the unique structural features of a large telomere repeat DNA complex (TRDC) of >20 kb generated by a simple PCR using (TTAGGG)(4) and (CCCTAA)(4) as both primers and templates. Although large, as determined by conventional agarose gel electrophoresis, the TRDC was found to consist of short single-stranded DNA telomere repeat units of between several hundred and 3000 bases, indicating that it is a non-covalent complex comprising short cohesive telomere repeat units. S1 nuclease digestion showed that the TRDC contains both single- and double-stranded portions stable enough to survive glycerol density gradient centrifugation, precipitation with ethanol and gel electrophoresis. Sedimentation analysis suggests that a part of the TRDC is non-linear and consists of a three-dimensional network structure. After treatment with Werner DNA helicase the TRDC dissociated into smaller fragments, provided that human replication protein A was present, indicating that: (i) the TRDC is a new substrate for the Werner syndrome helicase; (ii) the telomere repeat sequence re-anneals rapidly unless unwound single-stranded regions are protected by replication protein A; (iii) the TRDC may provide a new clue to understanding deleterious telomere-totelomere interactions that can lead to genomic instability. Some properties of the TRDC account for the extra-chromosomal telomere repeat (ECTR) DNA that exists in telomerase-negative immortalized cell lines and may be involved in maintaining telomeres.
Ohsugi
I
I
AGENE Research Institute, 200 Kajiwara, Kamakura, Kanagawa 247-0063, Japan.
Tokutake
Y
Y
Suzuki
N
N
Ide
T
T
Sugimoto
M
M
Furuichi
Y
Y
D002499:Centrifugation, Density Gradient; D004247:DNA / Q000737:chemistry*; D004265:DNA Helicases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005090:Exodeoxyribonucleases; D006801:Humans; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D053484:RecQ Helicases; D012091:Repetitive Sequences, Nucleic Acid; D051716:Replication Protein A; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D016615:Telomere; D014898:Werner Syndrome / Q000201:enzymology* / Q000235:genetics; D000071657:Werner Syndrome Helicase
D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D004247:DNA; D005090:Exodeoxyribonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase