Abstract 1110 Biochemical characterization of putative Mre11 homologs from Mimivirus and Marseillevirus

Mre11/Rad50 is a nuclease/ATPase protein complex, and their homologs are present ubiquitously in organisms from all domains of life as well as in viruses with double-stranded DNA genomes. This complex is an integral part of DNA repair machinery and is associated with double-stranded DNA break repair through homology/microhomology to maintain genomic integrity. Mutation in Mre11/Rad50 results in predisposition to cancer in humans while in lower organisms it increases their susceptibility towards DNA damage. Here, we report the characterization of Mre11 homologs from two amoeba-infecting viruses belonging to the Nucleocytoviricotaphylum, namely, Anacthamoeba polyphagaMimivirus (APMV) and Marseillevirus (MRSV). These viruses carry large double-stranded DNA genomes (1 Mbp, Mimivirus; 350 Kbp, Marseillevirus) and their replication occurs in the host cytoplasm in a nucleus-independent manner in organelle-like replication centres called "viral factories". The repertoire of DNA repair enzymes encoded in these viral genomes is hypothesized to play key roles in the maintenance of genomic integrity and aid replication inside viral factories. Previously, our lab had characterized a unique primase-polymerase as well as the base excision repair pathway operative in APMV. The current study was aimed at investigating the functional roles of giant viral putative Mre11 homologs. While APMV encodes a Mre11-Rad50 fusion protein, the activities are present on two polypeptides in the case of MRSV. In silico studies including sequence alignment and homology modelling and in vitro biochemical characterization of both wildtype recombinant Mre11 and its mutants were performed to investigate the Mre11 homologs. The in-silico studies showed a limited sequence similarity (< 30%) between the two Mre11 minimal functional domains suggesting sequence diversity of Mre11 of giant viruses, although overall structural similarity was observed. We expressed and purified the proteins and performed DNA binding assays confirming the binding of the Mre11 proteins to single as well as double-stranded DNA. Furthermore, nuclease assays revealed a contrasting feature where APMV Mre11 domain showed canonical single-stranded and double-stranded nuclease activities, while MRSV Mre11 protein showed only the single-stranded nuclease activity. The active site mutant lost the nuclease activity as expected. We also observed that the co-factor requirement of APMV Mre11 domain is similar to other Mre11 proteins reported. Studies on the effect of knock-out of Mre11 from Marseillevirus and knockout of Mre11 domain from the Mre11/Rad50 fusion protein of APMV are currently underway and should help in further delineating their roles. In conclusion, our study characterizes Mre11 homologs from giant viruses and suggests a functional difference between the two homologs which share broad structural similarities but possess two distinct activities.