Functional validation of activity and stability of BER mutants
Rm 2091210 will be BSLI lab space, Rm 327 is BSl2-certified
Conditions for ihe Approval of DOST-BC Ref. No. 201&0297: Functional Analysis of non-synonymous single nucleotide polymorphisms in DNA base excision repair enzymes
a) All activities shall be conducted at the lnstitute of Chemistry - Research Building Rooms 327 and 209-210 of the University of Philippines Diliman in Quezon City
b) The proponent shall adhere as closely as possible to the schedule of activities refiected in the submitted Gantt chart.
c) Any modifications in the schedule of activities shall be made with the concurrence of the UPD-lBC and the DOST-BC
d) A biosafety contingency plan shall be submitted before the conduct of the experiment.
e) The proponent shall inform which activities would require the presence of the UPD-IBC and DOST-BC.
f) The proponent shall ensure that only the DOST-BC authorized personnel are allowed inside the experimental facilities.
g) Any change in personnel allowed access to the project facilities, the proponent, thru the IBC is expected to submit the revised version of list of authorized personnel (Annex 7) along with the CV of the new project personnel in accordance with new CV format (Project Personnel Data Sheet)
h) The DOST-BC should be informed in advance of any visitations by unauthorized persons.
i) The DOST-BC shall be informed immediately of any intrusion by unauthorized persons.
j) The proponent shall ensure that stray anrmals are excluded from the experimental facilities while tests are being conducted.
k) ln case of undue destruction of experimental materials resulting from unauthorized entry of personnel or breach of containment of experimental facilities, the proponent shall implement measures to prevent the inadvertent escape of any viable material within the facility.
l) The proponent and the supervising IBC shall be held accountable for the undue destruction of the experimental materials and the consequences that their inadverient escape may cause to the surrounding environment.
m) All viable materials within the experiment area shall be accounted for.
n) The proponent shall strictly observe proper disposal procedures for all materials.
o) Movement of all materials will be done in compliance with all relevant biosafety and phytosanitary requirements of the Philippines.
p) Any additional requirement that the DOST-BC may impose, as necessary, for the duration of the experiment shall be complied with.
q) The proponent shall submit through the IBC a completion report within 90 days after completion of the experiment.
r) The IBC shall submit to the DOST-BC a reporl on the completion of this project, in the prescribed format.
This is a research proposal submitted to DOST-PCHRD that requires a biosafety clearance prior to proposal review, hence we are submitting this application. The proposed work involves recombinant expression of human DNA repair genes and their mutants. Genes (sequences obtained from NCBI EST database) will be synthesized and commercially purchased and will be cloned in commercially available bacterial protein expression cells (i.e., E. coli BL21 DE3). Recombinant E. coli will be harvested and lysed to extract the desired protein products. As per recommendation of the UPD-IBC, area where cloning will be performed (biosafety cabinet, bench space, etc) in my lab needs to be enclosed. ln the meantime, all proposed cloning work will be performed in Rm 327, a BSL2-certified lab, until such time that my lab, Rm 209/210 is deemed BSL1 compliant.
Constant exposure to environmental stress such as radiation and oxiding agents ultimately lead to the formation of abasic sites in DNA. To counter the mutagenic and cytotoxic effects of such DNA lesions, these sites are immediately sequestered and repaired by multiple, highly coordinated DNA repair enzymes and accessory proteins in the Base Excision Repair (BER) pathway. A common hallmark of several diseases, including cancer and neurodegeneration, is impaired DNA damage response mechanisms. Genetic variation arising from single nucleotide polymorphisms (SNPs) that result to amino acid substitutions of BER gene products can have important implications in the efficiency of DNA repair enzymes. While it has become almost routine to determine these SNPs through next-generation DNA sequencing methods, the actual effect (whether detrimental or actually non-threatening) of the mutations on the protein's activity still needs to be determined and functionally validated. We will identify variants in 10 DNA repair proteins involved in short-patch and long-patch BER by searching the Expressed Sequence Tag (EST) and SNP databases. Based on available 3D structures, we will perform in silico modeling, predict the functional effects of these enzyme variants and provide a struciural basis for impaired DNA repair activity, (in particular, examining effects on substrate DNA binding, catalytic function, post-translational modifications, and protein-protein interactions). A subset of these variants (3-5 mutants per BER protein) will be chosen for recombinant protein expression and purification and will be subjected to functional assays. Knowledge of the actual functional impairment of DNA repair protein mutations arising from SNPs will have wide implications in radiation-based chemotherapies. This will form the basis for diagnostics and pre-screening of patients with impaired DNA repair machinery.
Public Information Sheet
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