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MECHANISM OF HUMAN NONHOMOLOGOUS DNA END JOINING
by
Jiafeng Gu
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)
August 2009
Copyright 2009 Jiafeng Gu
Object Description
| Title | Mechanism of human nonhomologous DNA end joining |
| Author | Gu, Jiafeng |
| Author email | jiafengg@usc.edu; porter_gu@yahoo.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Molecular Biology |
| School | College of Letters, Arts and Sciences |
| Date defended/completed | 2009-06-01 |
| Date submitted | 2009 |
| Restricted until | Unrestricted |
| Date published | 2009-07-15 |
| Advisor (committee chair) | Lieber, Michael |
| Advisor (committee member) |
Goodman, Steven Chen, Xiaojiang Aparicio, Oscar Zandi, Ebrahim |
| Abstract | DNA double-strand breaks (DSBs) represent the most deleterious form of DNA damage, as both of the DNA strands are broken. In mammalian cells, DSBs are repaired predominantly by nonhomologous DNA end joining (NHEJ) pathway. NHEJ functions throughout the cell cycle to repair such lesions. Defects in NHEJ result in marked sensitivity to ionizing radiation and ablation of lymphocytes, which rely on NHEJ to complete the rearrangement of antigen-receptor genes. NHEJ is typically imprecise, a characteristic that is useful for immune diversification in lymphocytes, but which might also contribute to some of the genetic changes that underlie cancer and aging. To further understand the mechanism of human nonhomologous DNA end joining pathway, we performed in vitro biochemical reconstitution assay. Here, we present some distinctive features of polymerase mu and the ligase complex, XRCC4:DNA ligase IV:XLF.; Polymerase mu has both template dependent and template independent addition capabilities on DSB end substrate under physiological conditions. Template independent addition by polymerase mu prefers to add pyrimidines onto the DNA ends. In addition, template independent addition onto the 3' overhang of the DNA substrates by polymerase mu provides terminal microhomology for ligation by Ku and XRCC4:DNA ligase IV.; XRCC4 and DNA ligase IV form a complex that is essential for the repair of all double-strand DNA breaks by the NHEJ pathway in eukaryotes. We find here that, in the absence of processing factors, XRCC4:DNA ligase IV can ligate two double-strand DNA ends that have fully incompatible short 3' overhang configurations with no potential for base pairing. Moreover, at DNA ends that share 1-4 annealed base pairs, XRCC4:DNA ligase IV can ligate across gaps of 1 nucleotide. XLF stimulates the joining of both incompatible DNA ends and compatible DNA ends at physiological concentrations of Mg2+. Hence, the remarkable flexibility of the ligase complex is paramount in nonhomologous DNA end joining. These observations provide an explanation for several in vivo observations that were difficult to understand previously. Furthermore, those unusual ligation capabilities of XRCC4:DNA ligase IV:XLF complex may provide useful applications in recombinant DNA technology. |
| Keyword | biochemical reconstitution; cancer treatment; DNA double strand break; DNA repair and recombination; nonhomologous DNA end joining |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m2366 |
| Rights | Gu, Jiafeng |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-Gu-3003 |
| Archival file | uscthesesreloadpub_Volume26/etd-Gu-3003.pdf |
Description
| Title | Page 1 |
| Full text | MECHANISM OF HUMAN NONHOMOLOGOUS DNA END JOINING by Jiafeng Gu A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (MOLECULAR BIOLOGY) August 2009 Copyright 2009 Jiafeng Gu |
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