Biochemical and genetic studies on origin recognition and DNA synthesis from the mesophilic archaeon Methanosarcina acetivorans.

摘要

DNA replication starts from specific regions, known as replication origins in the genome. The process of initiating DNA replication needs a group of proteins that specifically assemble on DNA origins in a precisely determined manner. In eukaryotes the origin recognition complex (ORC) binds to origins of replication to initiate replication. M. acetivorans possesses two genes encoding Cdc6-like proteins, Cdc6-1 and Cdc6-2, with homology to both ORC and Cdc6 proteins. Both the biochemical and genetic studies for Cdc6-1 and Cdc6-2 were performed to study their roles in the initiation of replication. Our gel filtration analysis suggested that Cdc6-1 has higher affinity for origin-specific dsDNA than random dsDNA, but Cdc6-2 shows low binding affinity to both origin-specific and random dsDNA. Our genetic studies demonstrated that Cdc6-1 is essential but Cdc6-2 is not essential to M. acetivorans . According to both the biochemical and genetic data, Cdc6-1 is suspected to be the origin recognition protein in M. acetivorans.;Most organisms possess several DNA polymerase in charge of different functions such as replication, repair, and recombination. There are genes encoding four DNA polymerases, PolBI, PolD, PolX, and DinB in M. acetivorans . However, the replicative DNA polymerases in Euryarchaeota are not well understood to date. Our genetic studies showed that PolBI is essential to M. acetivorans, but DP1, PolX, DinB are not essential. It suggests that PolBI is the primary replicative DNA polymerase in M. acetivorans; other DNA polymerases may play important roles in recombination and repair.;In our study, we characterized DinB and found that DinB was able to synthesize through abasic DNA and cyclobutane pyrimidine dimers, and these activities were stimulated by the cognate PCNA. Fascinatingly, unlike most translesion DNA polymerases previously described, MacDinB synthesized an unusually long product (∼7.2 kb) in the presence of the sliding clamp. A PCNA-interacting peptide (PIP) box located at the C-terminus of MacDinB was shown to facilitate interaction with the sliding clamp. Furthermore, individual mutations at different positions in the PIP-box led to identification of residues that are critical to PCNA/DinB interactions. Nevertheless, the biochemical analysis described here for DinB provides important insights into the properties, potential, and diversity of the Y-family of DNA polymerases.