{"id":734,"date":"2024-10-12T04:14:01","date_gmt":"2024-10-12T04:14:01","guid":{"rendered":"http:\/\/cetaitdemain.org\/?p=734"},"modified":"2024-10-12T04:14:01","modified_gmt":"2024-10-12T04:14:01","slug":"265-13221c13230-pubmed-google-scholar-36","status":"publish","type":"post","link":"https:\/\/cetaitdemain.org\/?p=734","title":{"rendered":"\ufeff265, 13221C13230 [PubMed] [Google Scholar] 36"},"content":{"rendered":"

\ufeff265, 13221C13230 [PubMed] [Google Scholar] 36. of DNA polymerase -primase. or knockdown in mammalian cell lines resulted in G-overhang extension, DNA damage response, and sporadic telomere loss, XL388 whereas mutant plants showed severe telomere length deregulation phenotype and growth defects (5, 6). Together, these results demonstrated the presence of a conserved mechanism of telomere end protection from yeast to human (5C8). RPA is also known to bind to the telomeric 3-overhang in the S phase and to be involved in the regulation of telomere length (9C12). It is also required for DNA damage checkpoint activation at deprotected telomeres (13). Another known telomere-associated SSB is POT1 (protection of telomeres 1), which is conserved in a wide range of eukaryotes, including fission yeast, mammals, and plants, and binds with high affinity to G-rich telomeric repeat sequences. It is believed that POT1 precludes RPA from binding to the G-overhangs and activating the DNA damage signaling pathways (14). Interestingly, POT1 does not seem to compete with CST in binding to telomeres, and the two are redundantly required to prevent chromosomal ends from being recognized as DNA damage (5). Taken together, at least three kinds of SSBs can bind to chromosomal ends depending on the situation. Although the simplest view is that CST and POT1 protect telomeres by antagonistically excluding RPA from telomeres, some DNA damage responses may be required to form the appropriate telomere structures during and\/or after telomere replication (15). Thus, it is necessary to know how the different SSBs are coordinately targeted and function at a defined site to understand not only telomere biology but also other biological processes involving multiple SSBs. It has been reported that scStn1 interacts physically and genetically with the regulatory subunit of DNA polymerase , raising the possibility that Stn1 regulates the lagging DNA synthesis at telomeres (8, 16). In parallel with our identification of mammalian CST, another group reported that AAF-132 and AAF-44, which had been identified as mouse DNA polymerase -primase accessory proteins, regulate DNA replication in mammalian cells (17). Because AAF-132 and AAF-44 were found to be identical to Ctc1 and Stn1, respectively, it is important to clarify whether or not mammalian CST plays a role in the telomeric C-strand replication by DNA polymerase -primase. Although AAFs are suggested to XL388 be general DNA replication factors, our recent study challenged this idea by showing that endogenous human STN1 (hStn1) did not co-localize with DNA replication foci (5). It is still open to debate, however, when and where CST (AAFs) functions in cells. To investigate XL388<\/a> these issues further, we utilized egg extracts because they serve as excellent DNA replication model systems (18). egg extracts are cell-free systems that can be easily manipulated by immunodepleting the proteins of interest or adding various types of reagents. Unlike systems that are reconstituted with purified proteins and defined chemicals, egg extracts include essentially all factors that support early embryonic development and therefore faithfully recapitulate cellular events, including cell cycle progression. We describe herein the identification of CST and its involvement in priming DNA PRKD2<\/a> synthesis on ssDNA template in the egg extracts. Our data also showed that xCST is not an absolute requirement for chromosomal DNA replication. Our results are consistent with the hypothesis that CST is involved in the lagging strand synthesis in concert with DNA polymerase -primase at telomeres, in addition to its protective function. EXPERIMENTAL PROCEDURES Identification and Cloning of Xenopus laevis Ctc1, Stn1, and Ten1 The expressed sequence tag data base, Xenbase, was searched for transcripts potentially encoding Ctc1, Stn1, and Ten1, using the amino acid sequences of human homologs as queries. Full-length cDNAs were obtained by conventional RT-PCR techniques using total RNA derived from unfertilized eggs. The alignments of the and human amino acid sequences were performed using the ClustalW program on the website of DNA Data Bank XL388 of Japan. Antibodies and Recombinant Proteins We immunized two rabbits with full-length xStn1 recombinant protein (N-terminally His10-tagged, expressed in BL21-Codonplus (DE3) and purified using Ni-NTA-agarose (Qiagen)) to raise anti-xStn1 antibodies and obtained two lots of antisera, KU003 and KU004. IgG was affinity-purified using the antigen-blotted membrane..<\/p>\n","protected":false},"excerpt":{"rendered":"

\ufeff265, 13221C13230 [PubMed] [Google Scholar] 36. of DNA polymerase -primase. or knockdown in mammalian cell lines resulted in G-overhang extension, DNA damage response, and sporadic telomere loss, XL388 whereas mutant plants showed severe telomere length deregulation phenotype and growth defects (5, 6). Together, these results demonstrated the presence of a conserved mechanism of telomere end …<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[51],"tags":[],"class_list":["post-734","post","type-post","status-publish","format-standard","hentry","category-pkb","entry entry-center"],"_links":{"self":[{"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=\/wp\/v2\/posts\/734","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=734"}],"version-history":[{"count":1,"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=\/wp\/v2\/posts\/734\/revisions"}],"predecessor-version":[{"id":735,"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=\/wp\/v2\/posts\/734\/revisions\/735"}],"wp:attachment":[{"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=734"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=734"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/cetaitdemain.org\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=734"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}