Human REV3L: Expression and protein interaction studies

摘要: Human REV3L: Expression and Protein Interaction StudiesGregory N. Gan, Ph.D.University of Pittsburgh, 2007REV3L is a specialized DNA polymerase essential for damage-induced mutagenesis the ability cells to tolerate damage. Our understanding REV3L biochemistry stems predominantly from studies done with budding yeast homolog, Rev3. Yeast zeta consists two proteins, Rev3, catalytic subunit, Rev7, an accessory factor which enhances activity in vitro. Rev1 acts as scaffold by associating Polymerase its translesion bypass on mismatch primer template. Because large size mammalian cDNA (10.6 kbp) protein (353 kDa), work this field has focused solely functional genetic associated disruption or knockdown gene. Loss causes embryonic lethality mice leads progressive chromosomal instability Rev3L disrupted cell lines. In developing mouse embryo, transcript found all tissues. However, expression pattern at cellular level adult not been examined. Determining interactions will provide better how functions molecular level. addition, elucidating expressed regulated indicate what role it may play tissues organism why life. order study human biochemistry, project cloning, expressing, purifying detecting full-length protein. was hypothesized interact REV1 and/or REV7 based knowledge about te homologs. Furthermore, using lacZ model, organs containing proliferative characterized. It that highly tissue require REV3L. First, results immunoprecipitation demonstrated interacts REV1, but does damage independent fashion. Preliminary analysis deletion mutants indicates C-terminal domain required protein-protein interaction. Secondly, are ubiquitinated fashion covalent modification REV1-REV3L Finally, highest testis, cardiac smooth musculature lung intestines low lymphoid These sites suggest be important oxidative compared tissue. summary, dissertation provides insight REV3L's interaction REV7; their post-translational modification, tissue-specific mouse.