Preliminary Study On The Function Of Two Human Mutation-related Genes HMMS2 And CROC-1 By Antisense Strategy

Genetic instability is a main phenomenon in inherited diseases and cancers and may play a major role in their genesis and can be induced by environmental chemical carcinogenic agents. Our previous results showed that the chemical carcinogen-MNNG can induce the genetic instability which is characterized as nontargeted mutagenesis in mammalian cells and we proposed that signal transductions were activated through DNA damage pathway and non-DNA damage pathway to result in alteration of many genes expression when mammalian cells were treated with MNNG, and this led to the change of spectrums of DNA polymerases and several low fidelity DNA polymerases were employed in DNA replication, and thus the genetic instability was occurred in the treated cells.In recent years, it was proved that DNA damage tolerance is accomplished by DNA translesion synthesis(TLS). Since 1996, a lot of DNA polymerases involved in TLS were identified in prokaryotic and eukaryotic cells successively. This new kind of DNA polymerases can bypass DNA damages present in template strands in error-free and/or error-prone(mutagenic) ways and the majority of them have a low replication fidelity, lacking 3挆*5?proofreading function.MMS2 gene encoding product is a regulating factor involved in the errorfree TLS subpathway in S.cerevisiae. MMS2 gene encodes a ubiquitinconjugating enzyme-like protein(Ubc-like protein), and its amino acid sequence7and second and tertiary structures are similar to that of the ubiquitin-conjugating enzyme(UBC,E2). But unlike the ubiquitin-conjugating enzymes, Ubc-like protein does not appear to possess ubiquitin-conjugating activity because of lacking a critical cysteine residue in its active center. The ubiquitin-conjugating pathway involves in numerous metabolic processes in cells, including DNA damage repair, cell cycle control, stress responses and so on. The Ubc-like protein may play roles in E2 dominant negative variant form in vivo and may be a new regulator in the ubiquitinaton process of intracellular proteins. MMS2 gene encoding product mediates the two error-free TLS subpathways in which RAD5 and RAD3O genes are the representatives respectively in S.cerevisiae. It was found that the yeast mms2 null mutant was moderately sensitive to methyl methanesulfonate(MMS) and UV, and its spontaneous mutation frequency was dramatically increased. The mms2 mutant did not disrupt the REV3-dependent error-prone TLS subpathway and it displayed a REV3-dependent mutator phenotype.hMM~S2 and GROG-i are two newly identified human homologues of the yeast MMS2 gene and their encoding products are also Ubc-like proteins. hMms2 and Croc- 1 proteins share 50% and 75% amino acid sequence similarity with the yeast Mms2 protein respectively. It was suggested that CROC-1 gene may involve in tumorigenesis in human cells through its cell differentiation and cell cycle progress regulatory activity and may be considered as an oncogene in human cells. No direct research information about IiMMS2 gene in human cells was obtained at present. But it was found that hMMS2 and GROG-i genes were able to functionally complementary to the mms2 defects in yeast with regard to sensitivity to alkylating agent MMS and UV and spontaneous mutagenesis. And it was also discovered that both hMMS2 and MMS2 genes were able to transactivate a c-fos-CAT reporter gene in Rat-I cells in a transient cotransfection assay. Thus, it was inferred that the function of hMMS2 and GROG1 genes may be similar to that of the yeast MMS2 gene.Based on the theory for the mechanism of genetic instability we suggested and the function of yeast homologous gene MMS2, this research was proposed and its key aim is to study the relationship between the function of hMMS2 and GROG-i genes and the MNNG-induced genetic instability in human cells which is characterized as nontargeted mutagenesis. Thus, two eukaryotic expression8recombinants which can express hMMS2 and GROG-i gene antisense RNAs respectively under the dexamethasone(DEX) inductio...