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Graduation date: 2003

Graduation date: 1999

Nonhomologous end-joining (NHEJ) is a major DNA double-strand break (DSB) repair pathway. NHEJ is initiated through DSB recognition by the DNA end-binding heterodimer, Ku, while end-joining is accomplished by the XRCC4-DNA ligase IV (X4L4) complex. This thesis reports that APLF (Aprataxin and Polynucleotide kinase-Like Factor), an endo/exonuclease with a forkhead-associated (FHA) domain and two unique zinc fingers (ZF), interacts with both Ku and X4L4. The APLF-X4L4 interaction is FHA- and phospho-dependent, and is mediated by CK2 phosphorylation of XRCC4 in vitro. APLF binds Ku independently of the FHA and ZF domains, and complexes with Ku at DNA ends. APLF undergoes ionizing radiation induced ATM-dependent hyperphosphorylation and ATM phosphorylates APLF in vitro. Downregulation of APLF is associated with defective NHEJ and impaired DSB repair kinetics. These results suggest that APLF is an ATM target that is involved in NHEJ and facilitates DSB repair, likely via interactions with Ku and X4L4.

A DNA palindrome is a sequence of DNA followed by an exact inverted copy of itself. Palindromes are associated with gross chromosomal instability in mammalian cells. This may be related to their ability to extrude a double-stranded cruciform structure. In mammalian cells, palindromes have been shown to undergo centre-directed rearrangements resulting in a central region of asymmetry. This process occurs via a mechanism termed â centre break palindrome revisionâ . In this thesis, I have investigated palindrome revision in mammalian cells using two existing assays. In the first, performed by transfection of an extrachromosomal palindromic dimer, I have shown that joining of palindrome-mediated double-strand breaks does not depend solely on NHEJ and instead relies heavily on an alternate end-joining pathway. Using the second assay, the Line78 mouse model which contains a 15.4kb transgenic palindrome, I have shown that small modifications near the centre of the palindrome prevent these centre-directed rearrangements possibly by inhibiting cruciform formation.

Nonhomologous end-joining (NHEJ) is a major DNA double-strand break (DSB) repair pathway. NHEJ is initiated through DSB recognition by the DNA end-binding heterodimer, Ku, while end-joining is accomplished by the XRCC4-DNA ligase IV (X4L4) complex. This thesis reports that APLF (Aprataxin and Polynucleotide kinase-Like Factor), an endo/exonuclease with a forkhead-associated (FHA) domain and two unique zinc fingers (ZF), interacts with both Ku and X4L4. The APLF-X4L4 interaction is FHA- and phospho-dependent, and is mediated by CK2 phosphorylation of XRCC4 in vitro. APLF binds Ku independently of the FHA and ZF domains, and complexes with Ku at DNA ends. APLF undergoes ionizing radiation induced ATM-dependent hyperphosphorylation and ATM phosphorylates APLF in vitro. Downregulation of APLF is associated with defective NHEJ and impaired DSB repair kinetics. These results suggest that APLF is an ATM target that is involved in NHEJ and facilitates DSB repair, likely via interactions with Ku and X4L4.

A DNA palindrome is a sequence of DNA followed by an exact inverted copy of itself. Palindromes are associated with gross chromosomal instability in mammalian cells. This may be related to their ability to extrude a double-stranded cruciform structure. In mammalian cells, palindromes have been shown to undergo centre-directed rearrangements resulting in a central region of asymmetry. This process occurs via a mechanism termed â centre break palindrome revisionâ . In this thesis, I have investigated palindrome revision in mammalian cells using two existing assays. In the first, performed by transfection of an extrachromosomal palindromic dimer, I have shown that joining of palindrome-mediated double-strand breaks does not depend solely on NHEJ and instead relies heavily on an alternate end-joining pathway. Using the second assay, the Line78 mouse model which contains a 15.4kb transgenic palindrome, I have shown that small modifications near the centre of the palindrome prevent these centre-directed rearrangements possibly by inhibiting cruciform formation.

The human Rad9A checkpoint protein is required for genomic stability and proper execution of the DNA damage checkpoint. Previous work has shown Rad9A to be the key member of a heterotrimeric toroidal structure known as the 911 complex, along with Hus1A and Rad1, which is similar in structure to PCNA. Recent literature suggests Rad9A is a novel oncogene, found to be upregulated in a number of cancers and high mRNA levels are thought to have a protective effect on tumour growth. This thesis describes two novel functions for the Rad9A protein. The first is as a facilitator for the nuclear translocation of the Claspin adaptor protein, required for successful Chk1-mediated checkpoint activation. The second is as part of a novel nuclear structure containing important members of the homologous recombination DNA repair pathway. Work described herein also confirms the existence of a Rad9A paralogue, Rad9B, that partially rescues defects associated with Rad9A-deficiency and is expressed in both tumour and undifferentiated embryonic stem cell lines. This work discusses these findings in the context of current literature and provides future experiments to continue investigations into the function of this vital checkpoint protein. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2010-03-26 12:02:41.304

No description available.

Poly(ADP-ribose) polymerase (PADPRP) is a nuclear enzyme with a well documented role in DNA repair. Inhibitors of PADPRP, (e.g. 3' substituted benzamides) potentiate the cytotoxicity of a wide range of antitumour drugs. The results presented in this thesis represent, to the best of my knowledge, the first comprehensive and quantitative assessment of the ability of a range of P ADPRP inhibitors to modulate the cellular responses to damaging agents. Two novel PADPRP inhibitors, 8-hydroxy-2-methyl quinazolin-4(3H)-one (NU1025) and 3,4 dihydro-5-methoxyisoquinolin-1-(2H)-one (PD 128763) were compared with two "classical" PADPRP inhibitors, 3-aminobenzamide (3AB) and benzamide (BZ). The relative potencies for 3AB, BZ, NU1025 and PD 128763 as PADPRP inhibitors in vitro were 1.0, ~1.0, ~43 and ~53 respectively. All compounds potentiated the growth inhibition and cytotoxicity of the monofunctional alkylating agent temozolomide (TM) in L1210 cells. For example, 10/-lM NUI025 and PD 128763 gave dose enhancement factors (DEF) of ~2 at 100/0 survival, whereas ImM 3AB and 0.5mM BZ where required to give similar DEF values. Cellular NADl- levels were depleted up to 50% by 1-2mM TM and this depletion was completely prevented by coincubation with 50-100µM PD 128763 and 1-3mM 3AB. TM induced DNA single strand break levels were increased in a concentration dependent manner by the P ADPRP inhibitors. Overall, the relative potencies for ability of the compounds to potentiate TM induced growth inhibition, cytotoxicity and DNA single strand breaks showed good correlation with those determined in an in vitro inhibition study, with both NU1025 and PD 128763 exhibiting ~60 fold increased inhibitory activity as compared to 3AB. The PADPRP inhibitors per se did not effect the growth or survival of the L 121 0 cells, nor increase DNA strand breakage. NAD+ is the substrate for PADPRP. A L1210 cell line made resistant to tiazofurin (TZ) utilising a step wise selection protocol was shown to be deficient in nicotinamide mononucleotide adenyl transferase (NMNAT) , the final enzyme required for NAD+ biosynthesis. The consequences of a reduced NMNAT activity (<3% of the parental line ) and an ~40% reduction in intracellular NAD+ levels were determined. The resistant cells showed an ~3 fold increased sensitivity to TM as compared to the parental cells. Upon coincubation with increasing concentrations of NU1025 in the presence of a fixed concentration of TM, growth inhibition was potentiated ~70 fold in the resistant cells but only ~10 fold in the parental cell line, demonstrating the reduced level of competition between NAD+ and NUI025 for PADPRP. However, DNA single strand breaks were increased in the resistant compared to the parental cell line only when NU1025 was coincubated with TM. In contrast, in the presence of the PADPRP inhibitors alone, equivalent growth inhibitory effects were observed in each of the cell lines, suggesting inhibition of PADPRP was not the cytotoxic effector. The ~40% NAD+ depletion observed could therefore suggest, that NAD+ levels in the resistant cells were reduced to, or near to the KmNAD+ for PADPRP.