DNA replication is a process which is common to all domains of life yet different replication mechanisms are seen among URLanisms. The mechanism by which Acanthamoeba polyphaga mimivirus (APMV) undergoes replication is not characterized. Presence of intergenic short terminal repeats reveals that Mimivirus genome assumes a unique Q shape during replication . The mechanism of replication on such a structure is not yet understood. With this bigger picture in mind, we have initiated the characterization of a putative primase that is probably involved in the DNA replicationand/or repair in Mimivirus. Sequence alignment of gp0577 the protein encoded by gene L537 with other primases reveals that it contains the motifs common to the superfamily Archaeo-Eukaryotic Primase (AEP) and it aligns with Primpols, a novel type of primase which has RNA and DNA polymerase activities. Our initial analysis revealed that gp0577 probably exists as a dimer in its native state. We also identified the presence of conserved motifs: DxD, sxH, h- and Zn binding motif . Using Dpni mediated site directed mutagenesis we have generated active site mutants for all motifs. Templates of defined sequence were utilized to investigate the mechanism of primer synthesis by gp0577. Previous studies have suggested that the rate-limiting step of primer synthesis occurs during primer initiation during the formation of the dinucleotide. Thus we checked the effect of different concentrations of rNTPs involved in initial dinucleotide synthesis on primer synthesis. Consistent with this idea, increasing the concentration of NTPs required for dinucleotide synthesis increased the rate of primer synthesis, whereas increasing the concentration of NTPs not involved in dinucleotide synthesis inhibited primer synthesis .
DNA replication is a process which is common to all domains of life yet different replication mechanisms are seen among URLanisms. The mechanism of replication on such a structure is not yet understood. With this bigger picture in mind, we have initiated the characterization involved in DNA replication .
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by which Acanthamoeba polyphaga mimivirus (APMV) undergoes replication is not characterized. Presence of intergenic short terminal repeats reveals that Mimivirus genome assumes a unique Q shape during replication . The mechanism of replication
of replication
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of a putative primase that is probably involved in the DNA replicationand/or repair in Mimivirus. Sequence alignment of gp0577 the protein encoded by gene L537 with other primases reveals that it contains the motifs common to the superfamily Archaeo-Eukaryotic Primase (AEP) and it aligns with Primpols, a novel type of primase which has RNA and DNA polymerase activities. Our initial analysis revealed that gp0577 probably exists as a dimer in its native state. We also identified the presence of conserved motifs: DxD, sxH, h- and Zn binding motif . Using Dpni mediated site directed mutagenesis we have generated active site mutants for all motifs. Templates of defined sequence were utilized to investigate the mechanism of primer synthesis by gp0577. Previous studies have suggested that the rate-limiting step of primer synthesis occurs during primer initiation during the formation of the dinucleotide. Thus we checked the effect of different concentrations of rNTPs involved in initial dinucleotide synthesis on primer synthesis. Consistent with this idea, increasing the concentration of NTPs required for dinucleotide synthesis increased the rate of primer synthesis, whereas increasing the concentration of NTPs not involved in dinucleotide synthesis inhibited primer synthesis
involved in DNA replication