Study On Thepost-Translational Modification Of Holliday Junctionresolvase Hje In Sulfolobus Islandicus

When the DNAdamageoccured, a variety of repair mechanismsinitiate to repair the damaged DNA.Homologous recombination (HR) is relatively accurate repair mechanismcompared with others, and plays an important role in double-strand DNA break repair and stalled replication forks restart.One of the key steps in HR is the resolution of Holliday Junctions (HJs), which can bemediated by Holliday Junctions resolvase. Now, two HJ resolvases Hje and Hjc have been identified from Sulfolobus solfataricus and S. islandicus, of which Hjc is conserved among the archaea, but Hje is only existedinCrenarchaeota.Our previous studies have found that on SDS-PAGE gelthe size of Hje protein expressed and purified in archaea was the same as that expressed and purified in E. coli, but the molecular size of Hje-C-His protein purified in Sulfolobus was 2 kDa larger than that expressed and purified in E. coli. In eukaryotes, the function of HJ resolvases is regulated by post-translational modification. For example, Genl and Yenl have phosphorylation and dephosphorylation modification. The modification affects the protein activity, size or localization. Microarray analysis of Hje-C-His over-expression strain revealed that the transcriptional level of SiRe 2056 that codes serine/threonine protein kinase rose by 3.25 times. So we hypothesized that HJ resolvase Hje in archaea may have the same modification.We also found thatthe growth of Hje-C-His over-expression strain was suppressed. So we speculate there are two possible phosphorylation sites at the C terminal of Hje:serine at 127(S127) and threonine at 134 (T134).First, we constructed Hje mutant over-expression strains in which these two siteswere mutated intoalanine (A) or glutamate (E) based on E233S strain, and thenmeasured the protein size and determined the growth. We found that when these two siteswere mutated into alanine, the size of Hje mutant was samller than Hje-C-his, and the growth of over-expression strainsrecovered to normal state.When wemutated these two sites into glutamate tomimicphosphorylationmodification in vivo, the growth of over-expression strains Sis/pSeSD-HjeT134E-C-his and Sis/pSeSD-HjeT134Ewere inhibited again, but the growth of Sis/pSeSD-HjeS127E-C-his and Sis/pSeSD-HjeS127E recovered to normal state.Biochemical chacterizationshowed that the activity of allthese Hje mutants were the same as the native Hje-C-his except that the activity of HjeS127E-C-his was decreased in vitro. This indirectly indicated the site T134 might be phosphorylated, which could regulate the resolvase activity of Hje.And S127was also an important site, we hypothesized that this sitemight be involved in the protein-protein interaction.Secondly, to eliminate the influence of C terminal his-tag, we constructed Sis/pSeSD-Hje-N-his over-expression strain. By measuring the protein size and growth curve, we found that the size of Hje-N-His was 16 kDa, which was the same as the native Hje, and the growth of strain was normal.After treated by phosphatase, Hje-N-His protein size remains same. As we know, the post-translational modification of Holliday Junction resolvase in eukaryotes only exists in specific cell cycle, so it can not be excluded that the modification of Hje occur under specific circumstance. So we hypothesized the phosphoyralation level of Hje-N-his was lower than Hje-C-his.Finally, to obtain more modified protein for mass spectrum analysis, we used DNA damage agent MMS to treat the cell according to the methods used in eukaryote. We found two different size Hje signalsappeared after treated by MMS.So we speculated that Hje might exist post-translational modification induced by MMS, and the modification would affect the protein size. Then we attemptedto enrichnative modified Hje through immune co-precipitation method to identify the modification types and find proteins interacted with Hje. However, because the amount of native Hje is too low, we failed to obtain the highly pure Hje. The immune co-precipitation method still needs to be improved to get modified Hje for mass spectrum analysis.