Many eukaryotic genes are members of multi-gene families due to gene duplications, which generate new copies that allow functional divergence. However, the relationship between
The MutS protein plays an important role in the DNA mismatch repair system. Mutations in the mutS gene can lead to genome instability and ultimately cell malfunction. Here we have established a method for identifying ...The MutS protein plays an important role in the DNA mismatch repair system. Mutations in the mutS gene can lead to genome instability and ultimately cell malfunction. Here we have established a method for identifying functional defective mutants of MutS by random mutation and rifampicin screening. Some novel functional sites in MutS were identified. The MutS mutant strains were analyzed using surface plasmon resonance, gel filtration and far-western methods to determine the molecular mechanisms behind the DNA mismatch repair function of MutS.展开更多
Functional deficiency of mismatch repair(MMR) system is one of the mechanisms of tumorigenesis.With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to...Functional deficiency of mismatch repair(MMR) system is one of the mechanisms of tumorigenesis.With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to build up a method to evaluate the functional status of the whole MMR system in the concerned tumors. The original ssDNA and dsDNA from wild type (wt) bacteriophage M13mp2 and its three derivates with mutation points in the lacZa gene have been used to construct two kinds of heteroduplex DNA molecules. One named del(2) has two bases deleted in the negative strand, the other has a G-G mismatch base pair in the negative strand too. Introducing this heteroduplex DNA into E. coli NR9162 (routS^-) without the MMR ability on the indicator plate with x-gal and IPTG,there are three kinds of plaques, mixture plaque as the charaeteristie phenotype of heteroduplex DNA, blue and clearplaques. If the cell extract is mismatch repair competent the percentage of the mixture plaque will decrease after incubation with these heteroduplex DNA, the repair efficiency is expressed in percentage as 100x (1 minus the ratio of percentages of mixture plaque obtained from the extract-treated sample and untreated samples), which can imply the functionai status of MMR system of certain samples. After large T-antigen-dependent SV-40 DNA replication assay cell extract from TK6, a human lymphoblastoid B-cell lymphoma cell line with MMR ability, and Lovo, a human colonic carcinoma cell line with MMR deficiency have incubated with these heteroduplex DNA. The repair efficiency of TK6 to del(2) is more than 60%, to G-G is more than 50%. The Lovo efficiency to del(2) is less than 10%, to G-G is less than 20%.Therefore, in this in vitro model used for functional analysis of mismatch repair of heteroduplex DNA as the repair target,TK6 can serve as the control for MMR proficiency and Lovo as the control for MMR deficiency. Using this model the tumor tissue from a case of hereditary nonpolyposis colorectal cancer (microsatellite instability high, MSI-H) was measured and lack of MMR ability was shown. And a case of sporadic rectal cancer (SRC) (mierosatellite stability, MSS) maintains MMR proficiency. The results indicate that the model is sensitive and dependable. It could be used to measure the funetion status of MMR system in tumor cell and/or tissues. This is a reliable method to investigate the mechanic of tumorigenesis. It is meaningful in the observation of the role of MMR in the initiation and progression of concerned tumors.展开更多
RadA is highly conserved in bacteria and belongs to the RecA/RadA/Rad51 protein su-perfamily found in bacteria,archaea and eukarya. In Archaea,it plays a critical role in homologous re-combination process due to its R...RadA is highly conserved in bacteria and belongs to the RecA/RadA/Rad51 protein su-perfamily found in bacteria,archaea and eukarya. In Archaea,it plays a critical role in homologous re-combination process due to its RecA-like function. In Escherichia coli,it takes part in conjugational recom-bination and DNA repair but is not as important as that of archaea. Using PSI-BLAST searches,we found that Deinococcus radiodurans RadA had a higher similarity to that of bacteria than archaea and eukarya. Disruption of radA gene in D. radiodurans resulted in a modestly decreased resistance to gamma radiation and ultraviolet,but had no effect on the resistance to hydrogen peroxide. Complementa-tion of the radA disruptant by both E. coli radA and D. radiodurans radA could fully restore its resistance to gamma radiation and ultraviolet irradiation. Further domain function analyses of D. radiodurans RadA showed that the absence of the zinc finger domain resulted in a slightly more sensitive phenotype to gamma and UV radiation than that of the radA mutant,while the absence of the Lon protease domain exhib-ited a slightly increased resistance to gamma and UV radiation. These data suggest that D. radiodurans RadA does play an important role in the DNA damage repair processes and its three different domains have different functions.展开更多
文摘Many eukaryotic genes are members of multi-gene families due to gene duplications, which generate new copies that allow functional divergence. However, the relationship between
基金supported by the National Natural Science Foundation of China (Grant No. 30670443)the Chinese Academy of Sciences (Grant Nos. KSCX1-YW-R-63, KSCX2-YW-G-017 and KZCX2-YW-420)
文摘The MutS protein plays an important role in the DNA mismatch repair system. Mutations in the mutS gene can lead to genome instability and ultimately cell malfunction. Here we have established a method for identifying functional defective mutants of MutS by random mutation and rifampicin screening. Some novel functional sites in MutS were identified. The MutS mutant strains were analyzed using surface plasmon resonance, gel filtration and far-western methods to determine the molecular mechanisms behind the DNA mismatch repair function of MutS.
文摘Functional deficiency of mismatch repair(MMR) system is one of the mechanisms of tumorigenesis.With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to build up a method to evaluate the functional status of the whole MMR system in the concerned tumors. The original ssDNA and dsDNA from wild type (wt) bacteriophage M13mp2 and its three derivates with mutation points in the lacZa gene have been used to construct two kinds of heteroduplex DNA molecules. One named del(2) has two bases deleted in the negative strand, the other has a G-G mismatch base pair in the negative strand too. Introducing this heteroduplex DNA into E. coli NR9162 (routS^-) without the MMR ability on the indicator plate with x-gal and IPTG,there are three kinds of plaques, mixture plaque as the charaeteristie phenotype of heteroduplex DNA, blue and clearplaques. If the cell extract is mismatch repair competent the percentage of the mixture plaque will decrease after incubation with these heteroduplex DNA, the repair efficiency is expressed in percentage as 100x (1 minus the ratio of percentages of mixture plaque obtained from the extract-treated sample and untreated samples), which can imply the functionai status of MMR system of certain samples. After large T-antigen-dependent SV-40 DNA replication assay cell extract from TK6, a human lymphoblastoid B-cell lymphoma cell line with MMR ability, and Lovo, a human colonic carcinoma cell line with MMR deficiency have incubated with these heteroduplex DNA. The repair efficiency of TK6 to del(2) is more than 60%, to G-G is more than 50%. The Lovo efficiency to del(2) is less than 10%, to G-G is less than 20%.Therefore, in this in vitro model used for functional analysis of mismatch repair of heteroduplex DNA as the repair target,TK6 can serve as the control for MMR proficiency and Lovo as the control for MMR deficiency. Using this model the tumor tissue from a case of hereditary nonpolyposis colorectal cancer (microsatellite instability high, MSI-H) was measured and lack of MMR ability was shown. And a case of sporadic rectal cancer (SRC) (mierosatellite stability, MSS) maintains MMR proficiency. The results indicate that the model is sensitive and dependable. It could be used to measure the funetion status of MMR system in tumor cell and/or tissues. This is a reliable method to investigate the mechanic of tumorigenesis. It is meaningful in the observation of the role of MMR in the initiation and progression of concerned tumors.
基金Acknowledgements The authors would like to thank Prof. Fu Junjie （Radiation Center of Zhejiang University） for his help in irradiation treatment. This work was supported by the National Basic Research Program of China （Grant No. 2004CB19604）, a grant for Distinguished Young Scientist of China （Grant No. 30425038） and the National Natural Science Foundation of China （Grant No. 30330020）.
文摘RadA is highly conserved in bacteria and belongs to the RecA/RadA/Rad51 protein su-perfamily found in bacteria,archaea and eukarya. In Archaea,it plays a critical role in homologous re-combination process due to its RecA-like function. In Escherichia coli,it takes part in conjugational recom-bination and DNA repair but is not as important as that of archaea. Using PSI-BLAST searches,we found that Deinococcus radiodurans RadA had a higher similarity to that of bacteria than archaea and eukarya. Disruption of radA gene in D. radiodurans resulted in a modestly decreased resistance to gamma radiation and ultraviolet,but had no effect on the resistance to hydrogen peroxide. Complementa-tion of the radA disruptant by both E. coli radA and D. radiodurans radA could fully restore its resistance to gamma radiation and ultraviolet irradiation. Further domain function analyses of D. radiodurans RadA showed that the absence of the zinc finger domain resulted in a slightly more sensitive phenotype to gamma and UV radiation than that of the radA mutant,while the absence of the Lon protease domain exhib-ited a slightly increased resistance to gamma and UV radiation. These data suggest that D. radiodurans RadA does play an important role in the DNA damage repair processes and its three different domains have different functions.
