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"uri": "/id/document/15233" } ] } } ], "eprint_status": "archive", "userid": 247, "dir": "disk0/00/00/53/28", "datestamp": "2012-08-30 18:06:33", "lastmod": "2019-11-08 18:07:20", "status_changed": "2012-08-30 18:06:33", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "email": "oro27@mit.edu", "id": "Gold-Daniel-A", "name": { "family": "Gold", "given": "Daniel A." }, "show_email": "NO" } ] }, "title": "Molecular Characterization of the Dbf4/Drf1-Dependent Kinase (DDK) and the DNA Replication Checkpoint Mediator Claspin in Xenopus Egg Extracts", "ispublished": "unpub", "full_text_status": "public", "keywords": "Replication; Checkpoint; S-phase; DNA damage; Cell Cycle; Initiation; Genomic Instability; Tumorigenesis; Cancer; Claspin; DDK; Cdc7; Drf1", "abstract": "
The integrity of DNA replication control and checkpoint mechanisms is essential for preventing tumorigenesis. DNA replication is initiated by the S-CDK and DDK kinases which mediate the unwinding of the replication fork. Genotoxic stress which specifically affects cells in S-phase is detected by the replication checkpoint. Replication blockages activate the ATR kinase which, in turn, activates the downstream effector kinase Chk1 through the mediator protein, Claspin. Chk1 facilitates the arrest of cell cycle progression and the inhibition of replication origin firing.
\r\n\r\nClaspin has two broadly defined roles, one to mediate Chk1 activation and the other as a component of the replication fork. We have endeavored to study a link between these two facets of Claspin function. Here, we show that Claspin associates with several core replication fork proteins in Xenopus egg extracts. We identified a replication fork-interacting domain on Claspin that associates with the replication fork proteins and is required for Claspin association with chromatin. However, chromatin binding-deficient Claspin proteins can still mediate Chk1 activation in Claspin-depleted extracts, albeit with reduced efficiency. Thus, the localization of Claspin to the replication fork is not required for mediation of Chk1 activation but it does potentiate this process.
\r\n\r\nAnother focus of this study, DDK, is composed of the catalytic subunit Cdc7 and one of two distinct adaptor proteins, Drf1 or Dbf4. Drf1 forms a stable, active complex with Cdc7, even after replication arrest in egg extracts. Accumulation of Drf1 on chromatin in the presence of replication blocks is dependent upon ATR and Claspin but not Chk1. We characterized Xenopus Claspin as a kinase substrate of DDK which forms a stable nuclear complex with Cdc7 and Drf1 under both arrested and unperturbed replication conditions. Moreover, we identified a region of Claspin required for association with DDK that lies within the Chk1-binding domain, which contains a series of repeat sequences. This DDK-associating region is the first, but not the second of these repeat sequences. Furthermore, we have identified two evolutionarily conserved residues within this region required for DDK interaction. Claspin mutant proteins unable to interact with DDK still bind to Chk1 and rescue Chk1 activation in Claspin-depleted extracts. Therefore, we conclude that DDK regulates a largely checkpoint-independent role of Claspin function.
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Genes Dev 11, 3365-3374.\r\nLin, S.Y., Li, K., Stewart, G.S., and Elledge, S.J. (2004). Human Claspin works with BRCA1 to both positively and negatively regulate cell proliferation. Proc Natl Acad Sci U S A 101, 6484-6489.\r\nLiu, Q., Guntuku, S., Cui, X.S., Matsuoka, S., Cortez, D., Tamai, K., Luo, G., Carattini-Rivera, S., DeMayo, F., Bradley, A., et al. (2000). Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev 14, 1448-1459.\r\nLiu, S., Bekker-Jensen, S., Mailand, N., Lukas, C., Bartek, J., and Lukas, J. (2006). Claspin operates downstream of TopBP1 to direct ATR signaling towards Chk1 activation. Mol Cell Biol 26, 6056-6064.\r\nLou, H., Komata, M., Katou, Y., Guan, Z., Reis, C.C., Budd, M., Shirahige, K., and Campbell, J.L. (2008). Mrc1 and DNA polymerase epsilon function together in linking DNA replication and the S phase checkpoint. Mol Cell 32, 106-117.\r\nLupardus, P.J., Byun, T., Yee, M.C., Hekmat-Nejad, M., and Cimprich, K.A. (2002). A requirement for replication in activation of the ATR-dependent DNA damage checkpoint. Genes Dev 16, 2327-2332.\r\nMahbubani, H.M., Chong, J.P., Chevalier, S., Thommes, P., and Blow, J.J. (1997). Cell cycle regulation of the replication licensing system: involvement of a Cdk-dependent inhibitor. J Cell Biol 136, 125-135.\r\nMailand, N., Bekker-Jensen, S., Bartek, J., and Lukas, J. (2006). Destruction of Claspin by SCFbetaTrCP restrains Chk1 activation and facilitates recovery fr" ] }, "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Gayle Hammer", "deposited_by": "Daniel Gold", "deposited_on": "2012-08-30 18:06:33", "doi": "10.7907/XG90-5475", "alt_title": { "items": [ "Molecular Characterization of the Dbf4 Drf1-Dependent Kinase (DDK) and the DNA Replication Checkpoint Mediator Claspin in Xenopus Egg Extracts" ] }, "divisions": { "items": [ "div_biol" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "dunphy@caltech.edu", "id": "Dunphy-W-G", "name": { "family": "Dunphy", "given": "William G." }, "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "jcampbel@caltech.edu", "id": "Campbell-J-L", "name": { "family": "Campbell", "given": "Judith L." }, "role": "chair" }, { "email": "dchan@caltech.edu", "id": "Chan-D-C", "name": { "family": "Chan", "given": "David C." }, "role": "member" }, { "email": "pws@caltech.edu", "id": "Sternberg-P-W", "name": { "family": "Sternberg", "given": "Paul W." }, "role": "member" }, { "email": "haybruce@caltech.edu", "id": "Hay-B-A", "name": { "family": "Hay", "given": "Bruce A." }, "role": "member" }, { "email": "dunphy@caltech.edu", "id": "Dunphy-W-G", "name": { "family": "Dunphy", "given": "William G." }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2009-09-23", "gradofc_approval_date": "2009-12-16", "review_status": "approved", "option_major": { "items": [ "biology" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. 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} } ], "eprint_status": "archive", "userid": 1807, "dir": "disk0/00/00/58/47", "datestamp": "2011-06-20 16:43:41", "lastmod": "2019-11-08 18:10:23", "status_changed": "2011-06-20 16:43:40", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "email": "juanse307@gmail.com", "id": "Ramirez-Lugo-Juan-S", "name": { "family": "Ram\u00edrez-Lugo", "given": "Juan S." }, "show_email": "NO" } ] }, "title": "The Activation of ATR in Response to Double-Stranded DNA Breaks", "ispublished": "unpub", "full_text_status": "public", "keywords": "DNA Damage, Double-Stranded DNA Breaks, DSB, ATR, CtIP, Checkpoint, Cancer", "abstract": "The cellular response to the presence of double-stranded DNA breaks (DSBs) is primarily mediated by the ATM protein kinase. A related kinase, ATR, regulates the responses to dysfunctional DNA replication and is also activated, in an ATM-dependent manner, when breaks occur during S-phase. The latter is achieved by the ability of ATM to interact with TopBP1, an inducer of ATR activity. Additionally, in Xenopus egg extracts, the Mre11-Rad50-Nbs1 (MRN) complex is required to bridge ATM and TopBP1 together. With our current work, we show that CtIP, a known MRN-interacting protein, is recruited to DSB-containing chromatin and interacts with TopBP1 in a damage-dependent manner. A region containing the first two BRCT repeats of TopBP1 is essential for this interaction. Furthermore, two distinct regions of CtIP participate in mediating the association between CtIP and TopBP1. The first region includes two putative ATM/ATR phosphorylation sites. Secondly, an MRN-binding region in the N-terminal region of CtIP is involved. In addition, the binding between CtIP and TopBP1 is diminished in Nbs1-depleted extracts and, reciprocally, the binding of Nbs1 to TopBP1 decreases in the absence of CtIP. This suggests the formation of a complex containing CtIP, TopBP1 and the MRN complex. When CtIP is removed from egg extracts, the levels of TopBP1 and Nbs1 in damaged nuclei are reduced, thereby compromising the activation of the damage response. Thus, CtIP interacts with TopBP1 in a damage-stimulated, MRN-dependent manner to mediate the activation of ATR in response to DSBs. \r\n
\r\nWe additionally explore the involvement of the chromatin remodeling ATPase ISWI in the responses to DNA damage. We find that ISWI associates with ATR, ATRIP, and TopBP1 on DNA in the presence of damage. In addition, ISWI is a substrate of both ATM and ATR in vitro. Furthermore, the activities of ATM and ATR stimulate an increase in the levels of ISWI on chromatin that contains DSBs. Finally, we assessed the role of ISWI in the activation of multiple damage responses in Xenopus egg extracts. Taken together, our work describes several previously uncharacterized features of ISWI with implications in the response to damaged and incompletely replicated DNA.\r\n
", "date": "2010", "date_type": "degree", "id_number": "CaltechTHESIS:05262010-155732939", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:05262010-155732939", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "funders": { "items": [ { "agency": "NIH", "grant_number": "F31-GM72087-01 A1" } ] }, "collection": "CaltechTHESIS", "reviewer": "Gayle Hammer", "deposited_by": "Juan Ramirez-Lugo", "deposited_on": "2011-06-20 16:43:41", "doi": "10.7907/EG9Z-0H60", "divisions": { "items": [ "div_biol" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "dunphy@its.caltech.edu", "id": "Dunphy-W-G", "name": { "family": "Dunphy", "given": "William G." }, "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "woldb@caltech.edu", "id": "Wold-B-J", "name": { "family": "Wold", "given": "Barbara J." }, "role": "chair" }, { "email": "pws@caltech.edu", "id": "Sternberg-P-W", "name": { "family": "Sternberg", "given": "Paul W." }, "role": "member" }, { "email": "jcampbel@caltech.edu", "id": "Campbell-J-L", "name": { "family": "Campbell", "given": "Judith L." }, "role": "member" }, { "email": "dunphy@its.caltech.edu", "id": "Dunphy-W-G", "name": { "family": "Dunphy", "given": "William G." }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2010-05-14", "gradofc_approval_date": "2010-05-27", "review_status": "approved", "option_major": { "items": [ "biology" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2010", "author_list": "Ram\u00edrez-Lugo, Juan S.", "advisor_list": "Dunphy, William G.", "comittee_list": "Wold, Barbara J.; Sternberg, Paul W.; et el." }, { "id": "https://thesis.library.caltech.edu/id/eprint/5489", "eprint_id": 5489, "rev_number": 49, "documents": [ { "id": "/id/document/9169", "doc_id": 9169, "rev_number": 7, "files": [ { "id": "/id/file/57604", "fileid": 57604, "datasetid": "document", "objectid": 9169, "filename": "Chapter_1.pdf", "mime_type": "application/pdf", "filesize": 206099, "mtime": "2012-12-26 03:20:41", "url": "/5489/3/Chapter_1.pdf" } ], "eprint_id": 5489, "pos": 3, "placement": 3, "mime_type": "application/pdf", "format": "application/pdf", "format_desc": "Chapter 1", "language": "en", "security": "public", "license": "other", "main": "Chapter_1.pdf", "media_duration": "0", 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"dir": "disk0/00/00/54/89", "datestamp": "2010-01-07 17:58:18", "lastmod": "2021-04-19 20:25:47", "status_changed": "2010-01-07 17:58:18", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "email": "kew2001@hotmail.com", "id": "Wawrousek-Karen-Elizabeth", "name": { "family": "Wawrousek", "given": "Karen Elizabeth" }, "show_email": "YES" } ] }, "title": "Contributions of Dna2 and the Tim/Tipin Complex to Genomic Stability", "ispublished": "unpub", "full_text_status": "public", "keywords": "DNA replication; DNA repair; double-strand break; Dna2; Timeless; Tipin", "abstract": "This thesis describes the essential roles of Dna2 and the Tim/Tipin complex in the maintenance of genomic stability. Dna2 participates in DNA replication and double-strand break repair by homologous recombination. Meanwhile, the Tim/Tipin complex is required for efficient checkpoint activation upon replication stress, which can be caused by stalled DNA replication forks.
\r\n\r\nWhile yeast genetics and experiments with purified proteins have revealed much about yeast Dna2, we chose to pursue characterization of metazoan Dna2 using Xenopus cell-free extracts. We show that binding of Dna2 to origins of replication is dependent upon formation of pre-replication complexes but independent of CDK2 activity. Upon initiation of DNA replication, Dna2 travels with replication forks. Physical interactions with Mcm10 and And-1, proteins involved in lagging strand DNA replication, are indicative of a role in replication of the lagging strand; this result is consistent with genetic results in yeast and in vitro biochemical experiments.
\r\n\r\nDna2 also participates in the response to double-strand breaks and accumulates on chromatin containing double-strand breaks. We show that Dna2 binds to free DNA ends after the Mre11-Rad50-Nbs1 complex and ATM, but before RPA. Dna2-depleted extracts exhibit delayed processing of DNA ends, indicating that other nucleases do not easily compensate for the lack of Dna2. Consistent with genetic results in yeast, we find that the Mre11-Rad50-Nbs1 protein complex is essential for the processing of free DNA ends, but inhibition of Mre11 nuclease activity only slows processing. This observation indicates that other nucleases, possibly Dna2, can compensate for loss of Mre11 nuclease activity. Despite the role of Dna2 in double-strand break processing, Dna2 is not required for checkpoint activation.
\r\n\r\nTimeless (Tim) and Tipin participate in the checkpoint response to stalled replication forks. We demonstrate here that Tim and Tipin form a complex, associate with chromatin in S phase, and physically interact with many proteins at the replication fork. Human cells lacking the Tim/Tipin complex do not exhibit robust checkpoint activation in response to stalled replication forks. Finally, we show that Tipin is also a target of both the ATR and Cdc7 kinases, which respond to stalled replication forks.
\r\n", "date": "2010", "date_type": "degree", "id_number": "CaltechTHESIS:01032010-225423037", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:01032010-225423037", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Gayle Hammer", "deposited_by": "Karen Wawrousek", "deposited_on": "2010-01-07 17:58:18", "doi": "10.7907/QYSH-WH15", "divisions": { "items": [ "div_biol" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "email": "dunphy@caltech.edu", "id": "Dunphy-W-G", "name": { "family": "Dunphy", "given": "William G." }, "role": "advisor" } ] }, "thesis_committee": { "items": [ { "email": "jcampbel@caltech.edu", "id": "Campbell-J-L", "name": { "family": "Campbell", "given": "Judith L." }, "role": "chair" }, { "email": "dcrees@caltech.edu", "id": "Rees-D-C", "name": { "family": "Rees", "given": "Douglas C." }, "role": "member" }, { "email": "pws@caltech.edu", "id": "Sternberg-P-W", "name": { "family": "Sternberg", "given": "Paul W." }, "role": "member" }, { "email": "dunphy@caltech.edu", "id": "Dunphy-W-G", "name": { "family": "Dunphy", "given": "William G." }, "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2009-11-20", "gradofc_approval_date": "2010-01-06", "thesis_awards": "Lucy Guernsey Service Award, 2008.", "review_status": "approved", "option_major": { "items": [ "biochem" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2010", "author_list": "Wawrousek, Karen Elizabeth", "advisor_list": "Dunphy, William G.", "comittee_list": "Campbell, Judith L.; Rees, Douglas C.; et el." }, { "id": "https://thesis.library.caltech.edu/id/eprint/481", "eprint_id": 481, "rev_number": 19, "documents": [ { "id": "/id/document/717", "doc_id": 717, "rev_number": 3, "files": [ { "id": "/id/file/4548", "fileid": 4548, "datasetid": "document", "objectid": 717, "filename": "Shou_w_2001.pdf", "mime_type": "application/pdf", "filesize": 17332280, "mtime": "2012-12-26 02:30:03", "url": "/481/1/Shou_w_2001.pdf" } ], "eprint_id": 481, "pos": 1, "mime_type": "application/pdf", "format": "application/pdf", "language": "en", "security": "public", "license": "other", "main": "Shou_w_2001.pdf", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "content": "final", "relation": { "items": [ { "type": "http://eprints.org/relation/hasVolatileVersion", "uri": "/id/document/17535" }, { "type": "http://eprints.org/relation/haspreviewThumbnailVersion", "uri": "/id/document/17535" }, { "type": "http://eprints.org/relation/hasVersion", "uri": "/id/document/17535" } ] } }, { "id": "/id/document/16486", "doc_id": 16486, "rev_number": 3, "files": [ { "id": "/id/file/200156", "fileid": 200156, "datasetid": "document", "objectid": 16486, "filename": "Shou_Wenying_2001.txt", "mime_type": "text/plain", "filesize": 1699, "mtime": "2016-08-22 21:14:06", "url": "/481/2/Shou_Wenying_2001.txt" } ], "eprint_id": 481, "pos": 2, "format": "text/plain", "language": "en", "security": "internal", "license": "other", "main": "Shou_Wenying_2001.txt", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "content": "release" }, { "id": "/id/document/17535", "doc_id": 17535, "rev_number": 2, "files": [ { "id": "/id/file/4546", "fileid": 4546, "datasetid": "document", "objectid": 17535, "filename": "preview.png", "mime_type": "image/png", "hash": "84e874108351cee652f640a03bdc3db7", "hash_type": "MD5", "filesize": 7564, "mtime": "2012-12-26 02:30:03", "url": "/481/3/preview.png" } ], "eprint_id": 481, "pos": 3, "placement": 3, "mime_type": "image/png", "format": "image/png", "language": "en", "security": "public", "license": "other", "main": "preview.png", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "relation": { "items": [ { "type": "http://eprints.org/relation/isVolatileVersionOf", "uri": "/id/document/717" }, { "type": "http://eprints.org/relation/ispreviewThumbnailVersionOf", "uri": "/id/document/717" }, { "type": "http://eprints.org/relation/isVersionOf", "uri": "/id/document/717" } ] } }, { "id": "/id/document/119943", "doc_id": 119943, "rev_number": 1, "files": [ { "id": "/id/file/365312", "fileid": 365312, "datasetid": "document", "objectid": 119943, "filename": "indexcodes.txt", "mime_type": "text/plain", "hash": "bf84be867eaaa596dc5ef7bd4c5e03dd", "hash_type": "MD5", "filesize": 829, "mtime": "2021-06-23 20:28:25", "url": "/481/4/indexcodes.txt" } ], "eprint_id": 481, "pos": 4, "placement": 4, "mime_type": "text/plain", "format": "other", "format_desc": "Generate index codes conversion from text/plain to indexcodes", "language": "en", "security": "internal", "license": "other", "main": "indexcodes.txt", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "relation": { "items": [ { "type": "http://eprints.org/relation/isVersionOf", "uri": "/id/document/16486" }, { "type": "http://eprints.org/relation/isVolatileVersionOf", "uri": "/id/document/16486" }, { "type": "http://eprints.org/relation/isIndexCodesVersionOf", "uri": "/id/document/16486" } ] } } ], "eprint_status": "archive", "userid": 2, "dir": "disk0/00/00/04/81", "datestamp": "2005-02-07", "lastmod": "2022-09-13 20:35:01", "status_changed": "2009-09-25 01:38:15", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "id": "Shou-Wenying", "name": { "family": "Shou", "given": "Wenying" }, "orcid": "0000-0001-5693-381X", "show_email": "NO" } ] }, "title": "Diverse Mechanisms of Regulating the Mitotic Cell Cycle", "ispublished": "unpub", "full_text_status": "public", "keywords": "(Biology)", "abstract": "The mitotic cell cycle - a process in which one cell divides into two - is carefully regulated in response to signals. Organisms as diverse as yeast and human all drive their cell cycles by turning cyclin-dependent kinases (Cdks) on and off. We have examined Cdk regulation in two systems. First, we isolated a relatively general Cdk inhibitor (CKI) p28Kix1 in Xenopus laevis. p28Kix1 binds to and directly inhibits multiple Cdks, and retards DNA replication and mitosis when added to Xenopus egg extracts. Remarkably, the protein level of p28Kix1 is dramatically upregulated around late gastrulation, suggesting that it is induced in response to a developmental signal and in turn functions to establish a somatic type of cell cycle. Second, we examined how Cdk inactivation is achieved during mitotic exit in the yeast S. cerevisiae. We found that mutants in at least six linkage groups bypassed the mitotic arrest in cdc15\u0394 cells. The net1-1 mutant was studied further. Net1 inhibits mitotic exit by inhibiting Cdc 14, an essential protein phosphatase, using two parallel mechanisms: by binding and inactivating Cdc14, and by tethering Cdc14 to the nucleolus. Correct orientation of the mitotic spindle activates Cdc15, which evicts Cdc14 from the nucleolus into the entire cell. Cdc14 subsequently inactivates Cdks by promoting cyclin degradation and CKI induction, and mitotic exit ensues. Unexpectedly, Netl also regulates the structure and function of the nucleolus: it tethers the transcriptional silencing protein Sir2 to the nucleolus, regulates proper localization of multiple nucleolar antigens and rDNA morphology, and directly binds to and stimulates the activity of RNA Pol I. In summary, we observe that different signals regulate cell cycle progression by controlling Cdk activity, that cell cycle regulators may play important roles in other biological processes, and that localization of a protein to a subcellular structure could imply that it is sequestered instead of employed there.", "date": "2001", "date_type": "degree", "id_number": "CaltechETD:etd-02032005-110205", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechETD:etd-02032005-110205", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Kathy Johnson", "deposited_by": "Imported from ETD-db", "deposited_on": "2005-02-07", "doi": "10.7907/TP2C-Q082", "divisions": { "items": [ "div_biol" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "id": "Dunphy-W-G", "name": { "family": "Dunphy", "given": "William G." }, "orcid": "0000-0001-7598-8939", "role": "advisor" } ] }, "thesis_committee": { "items": [ { "id": "Deshaies-R-J", "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "orcid": "0000-0002-3671-9354", "role": "chair" }, { "id": "Wold-B-J", "name": { "family": "Wold", "given": "Barbara J." }, "orcid": "0000-0003-3235-8130", "role": "member" }, { "id": "Sternberg-P-W", "name": { "family": "Sternberg", "given": "Paul W." }, "orcid": "0000-0002-7699-0173", "role": "member" }, { "id": "Meyerowitz-E-M", "name": { "family": "Meyerowitz", "given": "Elliot M." }, "orcid": "0000-0003-4798-5153", "role": "member" }, { "id": "Dunphy-W-G", "name": { "family": "Dunphy", "given": "William G." }, "orcid": "0000-0001-7598-8939", "role": "member" }, { "id": "Anderson-D-J", "name": { "family": "Anderson", "given": "David J." }, "orcid": "0000-0001-6175-3872", "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_submitted_date": "2005-02-03", "thesis_defense_date": "2001-05-16", "thesis_approved_date": "2005-02-07", "thesis_awards": "Lawrence L. and Audrey W. Ferguson Prize, 2001", "review_status": "approved", "option_major": { "items": [ "biology" ] }, "copyright_statement": "I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive\nlicense to archive and make accessible, under the conditions specified below,\nmy thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2001", "author_list": "Shou, Wenying", "advisor_list": "Dunphy, William G.", "comittee_list": "Deshaies, Raymond Joseph; Wold, Barbara J.; et el." }, { "id": "https://thesis.library.caltech.edu/id/eprint/8114", "eprint_id": 8114, "rev_number": 27, "documents": [ { "id": "/id/document/46429", "doc_id": 46429, "rev_number": 3, "files": [ { "id": "/id/file/136053", "fileid": 136053, "datasetid": "document", "objectid": 46429, "filename": "lyapina 2001.pdf", "mime_type": "application/pdf", "hash": "d2785f794d8dddb668c64e31d15d7ec5", "hash_type": "MD5", "filesize": 18625089, "mtime": "2014-03-07 17:30:25", "url": "/8114/1/lyapina 2001.pdf" } ], "eprint_id": 8114, "pos": 1, "placement": 1, "mime_type": "application/pdf", "format": "application/pdf", "language": "en", "security": "public", "license": "other", "main": "lyapina 2001.pdf", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "content": "final" }, { "id": "/id/document/46430", "doc_id": 46430, "rev_number": 4, "files": [ { "id": "/id/file/136056", "fileid": 136056, "datasetid": "document", "objectid": 46430, "filename": "lyapina 2001.zip", "mime_type": "application/x-zip", "hash": "8ffcf6f2f254d92996bc52d56559e8fd", "hash_type": "MD5", "filesize": 59337444, "mtime": "2014-03-07 17:30:50", "url": "/8114/2/lyapina 2001.zip" } ], "eprint_id": 8114, "pos": 2, "placement": 2, "mime_type": "application/x-zip", "format": "application/zip", "format_desc": "TIFF", "language": "en", "security": "internal", "license": "other", "main": "lyapina 2001.zip", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "content": "archival" }, { "id": "/id/document/46431", "doc_id": 46431, "rev_number": 3, "files": [ { "id": "/id/file/136059", "fileid": 136059, 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"/id/document/46429" } ] } }, { "id": "/id/document/128828", "doc_id": 128828, "rev_number": 1, "files": [ { "id": "/id/file/374264", "fileid": 374264, "datasetid": "document", "objectid": 128828, "filename": "indexcodes.txt", "mime_type": "text/plain", "hash": "a51afe07ed29fb5dc47c57f70f625287", "hash_type": "MD5", "filesize": 35967, "mtime": "2021-06-24 03:13:39", "url": "/8114/7/indexcodes.txt" } ], "eprint_id": 8114, "pos": 7, "placement": 7, "mime_type": "text/plain", "format": "other", "format_desc": "Generate index codes conversion from application/pdf to indexcodes", "language": "en", "security": "public", "license": "other", "main": "indexcodes.txt", "media_duration": "0", "media_aspect_ratio": "0", "media_sample_start": "0", "media_sample_stop": "0", "relation": { "items": [ { "type": "http://eprints.org/relation/isVersionOf", "uri": "/id/document/46429" }, { "type": "http://eprints.org/relation/isVolatileVersionOf", "uri": "/id/document/46429" }, { "type": "http://eprints.org/relation/isIndexCodesVersionOf", "uri": "/id/document/46429" } ] } } ], "eprint_status": "archive", "userid": 50, "dir": "disk0/00/00/81/14", "datestamp": "2014-03-07 17:55:51", "lastmod": "2022-09-13 20:20:42", "status_changed": "2014-03-07 17:55:51", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "id": "Lyapina-Svetlana-Anatol'Evna", "name": { "family": "Lyapina", "given": "Svetlana Anatol'Evna" }, "show_email": "NO" } ] }, "title": "Characterization of the Human SCF Ubiquitin Ligases - Structure, Function, and Regulation", "ispublished": "unpub", "full_text_status": "public", "keywords": "Biology", "abstract": "The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by\r\ncata lyzi ng ubiquitination of the S phase CDK inhibitor SIC1. SCF is composed of several\r\nevolutionarily conserved proteins, including ySKP1, CDC53 (Cullin), and the F-box protein\r\nCDC4. We isolated hSKP1 in a two-hybrid screen with hCUL1, the human homologue of\r\nCDC53. We showed that hCUL1 associates with hSKP1 in vivo and directly interacts with\r\nhSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-Iike particle. Moreover,\r\nhCUL1 complements the growth defect of yeast CDC53^(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin\r\nligase complexes with yeast SCF components. These data demonstrated that hCUL1 functions as\r\npart of an SCF ubiquitin ligase complex in human cells. However, purified human SCF\r\ncomplexes consisting of CUL1, SKP1, and SKP2 are inactive in vitro, suggesting that additional\r\nfactors are required.
\r\n\r\nSubsequently, mammalian SCF ubiquitin ligases were shown to regulate various\r\nphysiological processes by targeting important cellular regulators, like l\u0138B\u03b1, \u03b2-catenin, and p27,\r\nfor ubiquitin-dependent proteolysis by the 26S proteasome. Little, however, is known about the\r\nregulation of various SCF complexes. By using sequential immunoaffinity purification and mass\r\nspectrometry, we identified proteins that interact with human SCF components SKP2 and CUL1\r\nin vivo. Among them we identified two additional SCF subunits: HRT1, present in all SCF\r\ncomplexes, and CKS1, that binds to SKP2 and is likely to be a subunit of SCF5^(SKP2) complexes.\r\nSubsequent work by others demonstrated that these proteins are essential for SCF activity. We\r\nalso discovered that COP9 Signalosome (CSN), previously described in plants as a suppressor of\r\nphotomorphogenesis, associates with CUL1 and other SCF subunits in vivo. This interaction is\r\nevolutionarily conserved and is also observed with other Cullins, suggesting that all Cullin based\r\nubiquitin ligases are regulated by CSN. CSN regulates Cullin Neddylation presumably through CSNS/JAB1, a stochiometric Signalosome subunit and a putative deneddylating enzyme. This\r\nwork sheds light onto an intricate connection that exists between signal transduction pathways\r\nand protein degradation machinery inside the cell and sets stage for gaining further insights into\r\nregulation of protein degradation.
\r\n", "date": "2001", "date_type": "degree", "id_number": "CaltechTHESIS:03072014-090536401", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:03072014-090536401", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Tony Diaz", "deposited_on": "2014-03-07 17:55:51", "doi": "10.7907/apdy-3d30", "divisions": { "items": [ "div_biol" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "id": "Dunphy-W-G", "name": { "family": "Dunphy", "given": "William G." }, "orcid": "0000-0001-7598-8939", "role": "advisor" } ] }, "thesis_committee": { "items": [ { "id": "Deshaies-R-J", "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "orcid": "0000-0002-3671-9354", "role": "chair" }, { "id": "Campbell-J-L", "name": { "family": "Campbell", "given": "Judith L." }, "role": "member" }, { "id": "Dunphy-W-G", "name": { "family": "Dunphy", "given": "William G." }, "orcid": "0000-0001-7598-8939", "role": "member" }, { "id": "Varshavsky-A-J", "name": { "family": "Varshavsky", "given": "Alexander J." }, "orcid": "0000-0002-4011-258X", "role": "member" }, { "id": "Wold-B-J", "name": { "family": "Wold", "given": "Barbara J." }, "orcid": "0000-0003-3235-8130", "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2001-01-30", "review_status": "approved", "option_major": { "items": [ "biology" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. 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"/id/document/46436" }, { "type": "http://eprints.org/relation/isIndexCodesVersionOf", "uri": "/id/document/46436" } ] } } ], "eprint_status": "archive", "userid": 87, "dir": "disk0/00/00/81/15", "datestamp": "2014-03-07 22:33:56", "lastmod": "2022-09-13 18:36:49", "status_changed": "2014-03-07 22:33:56", "type": "thesis", "metadata_visibility": "show", "creators": { "items": [ { "id": "Chi-Yong", "name": { "family": "Chi", "given": "Yong" }, "show_email": "NO" } ] }, "title": "Negative Regulation of Transcription Factors by Srb10 Cyclin-Dependent Kinase", "ispublished": "unpub", "full_text_status": "public", "keywords": "Biology", "abstract": "The ubiquitin-dependent proteolytic pathway plays an important role in a broad\r\narray of cellular processes, inducting cell cycle control and transcription. Biochemical\r\nanalysis of the ubiquitination of Sic1, the B-type cyclin-dependent kinase (CDK)\r\ninhibitor in budding yeast helped to define a ubiquitin ligase complex named SCFcdc4 (for\r\nSkp1, Cdc53/cullin, F-box protein). We found that besides Sic1, the CDK inhibitor Far1\r\nand the replication initiation protein Cdc6 are also substrates of SCFcdc4 in vitro. A\r\ncommon feature in the ubiquitination of the cell cycle SCFcdc4 substrates is that they must\r\nbe phosphorylated by the major cell cycle CDK, Cdc28. Gcn4, a transcription activator\r\ninvolved in the general control of amino acid biosynthesis, is rapidly degraded in an\r\nSCFcdc4-dependent manner in vivo. We have focused on this substrate to investigate the\r\ngenerality of the SCFcdc4 pathway. Through biochemical fractionations, we found that the\r\nSrb10 CDK phosphorylates Gcn4 and thereby marks it for recognition by SCFcdc4\r\nubiquitin ligase. Srb10 is a physiological regulator of Gcn4 stability because both\r\nphosphorylation and turnover of Gcn4 are diminished in srb10 mutants. Furthermore, we\r\nfound that at least two different CDKs, Pho85 and Srb10, conspire to promote the rapid\r\ndegradation of Gcn4 in vivo. The multistress response transcriptional regulator Msn2 is\r\nalso a substrate for Srb10 and is hyperphosphorylated in an Srb10-dependent manner\r\nupon heat stress-induced translocation into the nucleus. Whereas Msn2 is cytoplasmic in\r\nresting wild type cells, its nuclear exclusion is partially compromised in srb10 mutant\r\ncells. Srb10 has been shown to repress a subset of genes in vivo, and has been proposed\r\nto inhibit transcription via phosphorylation of the C-terminal domain of RNA polymerase\r\nII. Our results suggest a general theme that Srb10 represses the transcription of specific\r\ngenes by directly antagonizing the transcriptional activators.", "date": "2001", "date_type": "degree", "id_number": "CaltechTHESIS:03072014-113238525", "refereed": "FALSE", "official_url": "https://resolver.caltech.edu/CaltechTHESIS:03072014-113238525", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "collection": "CaltechTHESIS", "reviewer": "Tony Diaz", "deposited_by": "Benjamin Perez", "deposited_on": "2014-03-07 22:33:56", "doi": "10.7907/tb1q-fz89", "divisions": { "items": [ "div_biol" ] }, "institution": "California Institute of Technology", "thesis_type": "phd", "thesis_advisor": { "items": [ { "id": "Dunphy-W-G", "name": { "family": "Dunphy", "given": "William G." }, "orcid": "0000-0001-7598-8939", "role": "advisor" } ] }, "thesis_committee": { "items": [ { "id": "Deshaies-R-J", "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "orcid": "0000-0002-3671-9354", "role": "chair" }, { "id": "Bjorkman-P-J", "name": { "family": "Bjorkman", "given": "Pamela J." }, "orcid": "0000-0002-2277-3990", "role": "member" }, { "id": "Campbell-J-L", "name": { "family": "Campbell", "given": "Judith L." }, "role": "member" }, { "id": "Dunphy-W-G", "name": { "family": "Dunphy", "given": "William G." }, "orcid": "0000-0001-7598-8939", "role": "member" }, { "id": "Wold-B-J", "name": { "family": "Wold", "given": "Barbara J." }, "orcid": "0000-0003-3235-8130", "role": "member" } ] }, "thesis_degree": "PHD", "thesis_degree_grantor": "California Institute of Technology", "thesis_defense_date": "2001-05-22", "review_status": "approved", "option_major": { "items": [ "biology" ] }, "copyright_statement": "Author's Rights Authorization: I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted here is the same as that approved by my advisory committee.\n\nI hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified under \"Thesis Availability\" in this submission, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation, or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.", "resource_type": "thesis", "pub_year": "2001", "author_list": "Chi, Yong", "advisor_list": "Dunphy, William G.", "comittee_list": "Deshaies, Raymond Joseph; Bjorkman, Pamela J.; et el." } ]