The Telomere Binding Protein Cdc13 and the Single-Stranded DNA Binding Protein RPA Protect Telomeric DNA from Resection by Exonucleases.
The telomere is present at the ends of all eukaryotic chromosomes and usually consists of repetitive TG-rich DNA that terminates in a single-stranded 3' TG extension and a 5' CA-rich recessed strand. A biochemical assay that allows the in vitro observation of exonuclease-catalyzed degradation (resection) of telomeres has been developed. The approach uses an oligodeoxynucleotide that folds to a stem-loop with a TG-rich double-stranded region and a 3' single-stranded extension, typical of telomeres. Cdc13, the major component of the telomere-specific CST complex, strongly protects the recessed strand from the 5'→3' exonuclease activity of the model exonuclease from bacteriophage λ. The isolated DNA binding domain of Cdc13 is less effective at shielding telomeres. Protection is specific, not being observed in control DNA lacking the specific TG-rich telomere sequence. RPA, the eukaryotic single-stranded DNA binding protein, also inhibits telomere resection. However, this protein is non-specific, equally hindering the degradation of non-telomere controls.
Institute for Cell and Molecular Biology, The University of Newcastle, Newcastle upon Tyne NE2 4HH, United Kingdom.
Matthew
M
Greetham
The Wistar Institute, Philadelphia, PA 19104, USA.
Emmanuel
E
Skordalakes
Institute for Cell and Molecular Biology, The University of Newcastle, Newcastle upon Tyne NE2 4HH, United Kingdom.
David
D
Lydall
Institute for Cell and Molecular Biology, The University of Newcastle, Newcastle upon Tyne NE2 4HH, United Kingdom. Electronic address: bernard.connolly@ncl.ac.uk.
Bernard A
BA
Connolly
D010582:Bacteriophage lambda / Q000201:enzymology*; D001483:Base Sequence; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D051716:Replication Protein A / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D016615:Telomere / Q000737:chemistry / Q000378:metabolism*; D034501:Telomere-Binding Proteins / Q000378:metabolism*
C452219:Cdc13 protein, S cerevisiae; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; C082728:RFA1 protein, S cerevisiae; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D034501:Telomere-Binding Proteins; D005090:Exodeoxyribonucleases
CST complex; Cdc13; RPA; exonuclease-catalyzed resection; telomere