This experience has provided me with insight into the logic behind genetic engineering and experiment design and given me greater aptitude in various laboratory procedures including RT-PCR (reverse transcriptase polymerase chain reaction), plasmid transfection and recovery, plasmid transformation and amplification, and RNA isolation. It was the creativity of the design-associated aspects of our project that I found most satiating. In fact, in order to keep up with the evolution of the construction design, I learneda good deal of interesting biology that I now find the most valuable aspect of my internship. Enthusiastic about the knowledge I have gained while spending my days at the Cooper lab, I feel compelled to write this web-page with a focus that emphasizes the progression of my understanding about TCR and single-lesion containing shuttle vectors while it describes the experiments and protocol optimizations I performed. AbstractOur goal is to design a shuttle vector that contains a unique, site-specific lesion in order to study transcription-coupled repair of human DNA.
In our system, the lesion is introduced by insertion of a synthesized 8-oxoguanine-containing oligomer into a pS189-derived plasmid at either of two locations: within the t-antigen (Tag) intron 400 bases beyond the ATG translation start codon, or at the end of the Tag, after the polyadenylation signal. The pS189 shuttle vector was modified to increase the transcription frequency of the Tag, prevent plasmid replication, and distinguish between Tag derived from SV40-transformed cells and that from the shuttle vector. Initial studies were undertaken to optimize the transfection conditions and also to verify the various plasmid alterations. Preliminary RT-PCR of mRNA harvested 24 hours after plasmid transfection has demonstrated that use of primers tuned to the Tag modifications do successfully distinguish plasmid from cellular RNA. Replication assays using methylation-sensitive endonucleases have verified the competence of engineered mutations in the SV40 ori in achieving preclusion of plasmid replication.
RT-PCR has also shown low amplification near the Bgl II site, suggesting its removal during the processing of mature mRNA. It will therefore be necessary to construct a new site for lesion insertion before the poly-adenylation signal. In conclusion, with the competency of the pS189-derived plasmids confirmed by RT-PCR, both the shuttle vector and the transfection protocol have been optimized for TCR studies, and we are ready to insert the 8-oxoG-containing oligomer. IntroductionToward maintenance of genomic integrity and to combat the mutations and genetic degradation associated with aging and cancer, several intricate and interconnected pathways exist for DNA repair.
These repair pathways include nucleotide excision repair (NER) and base excision repair (BER). Excision and repair of lesions on the transcribed strand (TS) occurs at a higher frequency than on the non-transcribed strand (NTS). This phenomenon called transcription-coupled repair (TCR) seems to be initiated by RNA polymerase II stalled at lesions or lesion-protein complexes. Lesions introduced by reactive oxygen species generated during cellular metabolism, including 8-oxoguanine (8-oxoG), are subject to TCR, even though they do not impede DNA polymerase during replication. If left unrepaired, 8-oxoG lesions can mispair with adenine and, upon replication, cause a guanine to thymine transversion with 50% frequency. Impedance of TCR results in a degenerative disease, Cockayne Syndrome (CS), characterized by postnatal developmental failure, neurological degeneration and early death.
Another disease, Xeroderma Pigmentosum (XP), involves the global malfunction of nucleotide-excision repair (NER) due to mutations in the XPA-XPG proteins. Previously, investigators introduced oxidative lesions in a random, stochastic manner, making the detailed kinetics of TCR difficult to assay. These limitations can be surpassed with the constructing of site-specific single-lesion containing shuttle vectors. Our Research GoalsTo study transcription-coupled repair of oxidative damage to DNA in various cell lines from Cockayne Syndrome (CS) and Xeroderma Pigmentosum (XP) patients, using a single-lesion containing shuttle vector. To further characterize both the requirements for XPD/XPB/XPG in TCR, and the behavior of RNA Polymerase near oxidative lesions. To further assess the degree to which lesions caused by oxidative damage (specifically