APLF is a forkhead associated (FHA) domain-containing protein with unique poly(ADP)-ribose (PAR)-binding zinc finger (PBZ) domains that are involved in the DNA damage response. The interaction of the APLF PBZ domains with PAR is essential for the rapid recruitment of APLF to sites of DNA double strand breaks (DSBs), while the FHA domain facilitates non-homologous end-joining. In response to ionizing radiation (IR), APLF is phosphorylated at Serine-116 (APLFS116), although the function of this post-translational modification has yet to be defined. Here we provide a detailed characterization of the IR-induced and ATM- dependent phosphorylation of endogenous APLF at Serine-116 in the context of DNA damage. We additionally examine a novel APLF FHA-dependent interaction with 53BP1 (p53 Binding protein 1). Together, we illustrate that APLFS116 phosphorylation is dependent upon both the tandem PBZ domains, as well as the FHA-domain, and that the depletion of either PARP3 or 53BP1, similarly affected APLFS116 phosphorylation. Furthermore, we show that DSB-repair was compromised in cells expressing the APLFS116A mutation. Collectively, our findings provide a detailed understanding of the molecular pathway that leads to the phosphorylation of APLF following DNA damage and suggest that APLFS116 phosphorylation facilitates APLF-dependent DSB repair.

No description available.

The project was to clone the gene encoding a nuclear enzyme poly-ADP-ribose polymerase (PARP) involved in the posttranslational modification of nuclear proteins. This modification is important in the regulation of various cellular processes such as cell differentiation, proliferation and in the molecular events involved in the recovery of cells from DNA damage. At the start of this project this enzyme had been well characterized in animal systems but had not as yet been explored in plants. Firstly, I showed the enzyme to be present in higher plant nuclei, by a series of experiments including enzyme assays and western blotting (probed with a polyclonal antibody specific to the protein). Molecular biology approaches were used to isolate the gene encoding the PARP enzyme. These techniques included screening of cDNA libraries, constructed in λZAPII, with both the polyclonal antibody and a gene probe (a restriction digest of a human PARP gene was performed and a 1.4Kb fragment containing the C-terminal region of the gene used as a probe). This identified a number of bacteriophages from which sequence information was obtained (by making and extracting phagemids). Subsequent translation of these nucleotide sequences revealed that one of the sequences (245941) obtained in this manner showed homology to the PARP protein. Degenerate oligonucleotide primers, designed to the conserved C-terminal region of the gene, were also used in an attempt to amplify a 375bp region of the gene thought to contain the PARP signature. PCR products of the expected size (375bp) were obtained from an SST cDNA library. These PCR products hybridized to the gene probe (mentioned earlier). Subsequent subcloning revealed multiple products of 375bp comigrating in the agarose gels. Two different sequences were obtained which were, upon translation, shown not be PARP-like sequence.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 1999. / Härtill 6 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on December 20, 2007) Includes bibliographical references.

Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 3 uppsatser.

Thesis (M.S.)--Bowling Green State University, 2006. / Document formatted into pages; contains ix, 154 p. : ill. Includes bibliographical references.

Unrepaired, or misrepaired, DNA damage can be carcinogenic or mutagenic; thus functional DNA damage repair pathways are essential for the safeguarding of the genome. SNM1B is a 5' to 3' exonuclease implicated in the repair of damaged DNA, particularly the repair of interstrand crosslinks. Genetic studies have identified SNPs in the SNM1B gene as related to cancer risk. One of these (rs11552449) encodes a single amino acid change, H61Y. This study shows that WT and H61Y SNM1B have comparable in vitro biochemical and biophysical characteristics. The structures of both WT and H61Y C-terminally truncated SNM1B (Δ-SNM1B) were solved to 2.8 and 3.1 Å, respectively, and reveal similar structural properties. The structure of WT Δ-SNM1B was also reported (to 1.8 Å) with two 2'-deoxy-5'- adenosine monophosphate molecules in the active site. The structure of SNM1B shows an accessible extended active site, which may facilitate the binding of a variety of non-canonical DNA substrates. Accordingly, in vitro, WT and H61Y SNM1B are able to exonucleolytically process a wide range of structurally diverse DNA substrates. By utilising SNM1B depleted cell lines, this study also shows that SNM1B is required for DNA repair in response to treatment with DNA-crosslinking genotoxic agents (including cisplatin and SJG-136). This study also identifies the novel double strand break repair factor, EXD2, as having intrinsic 3' to 5' exonuclease activity. EXD2 was shown to have enzymatic activity on a variety of substrates in vitro, including replication fork intermediates, 'nicked' or 'gapped' DNA duplexes, and RNA based substrates. Together with the cellular data this suggests a role for EXD2 in nucleolytically processing RNA or DNA-based intermediates in damage repair pathways.

ATM is a large and complex gene, identified as the recessive gene mutated in individuals with the childhood syndrome ataxia telangiectasia. While AT patients are rare and the disease has many severe symptoms, including cerebellar degeneration, immunodeficiency and a very high cancer risk, the relatively common carriers of one mutation are clinically normal. However, there is some evidence that these heterozygotes, too, have a high relative risk of cancer, especially breast cancer in women. The cellular radio sensitivity of carriers is intermediate between that of normal individuals and that of AT patients, and this may suggest the cells are predisposed to tumorigenic transformation. ATM is believed to be involved in DNA damage response, but the mechanisms by which it works are not fully understood. In this study, DNA from 412 Scottish women with breast cancers were screened for two ATM mutations, known to be relatively common in Celtic populations. In attempting to estimate the burden in Scotland of breast cancers in AT heterozygotes, it is shown here that even these two most common ATM mutations in the UK account for only a small proportion of all ATM mutations. None of these mutations were found, suggesting that these particular mutations do not confer a predisposition to breast cancer. Also presented are the results of various immunological studies. The ATM protein is shown to be localised to specific structures within the nucleus of a normal cell. It appears to be found in a number of different forms, varying in size and state of glycosylation. It sediments at a relatively low speed, suggesting that it is normally bound in a large protein complex. This work indicates that a large-scale screening study will be required to establish whether or not AT carriers are at an increased risk of cancer, and reveals that the ATM product may be present in more than one form, depending on whether the cell' stimulated to divide.