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A structure-specific DNA endonuclease is enriched in kinetoplasts purified from Crithidia fasciculata
1Molecular Biology Institute and Department of Molecular, Cell and Developmental Biology, 611 Circle Drive East, University of California Los Angeles, Los Angeles, CA 90095-1570, USA.
Abstract:
The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata consists of minicircles and maxicircles topologically interlocked in a single network per cell. Individual minicircles replicate unidirectionally from either of two replication origins located 180 degrees apart on the minicircle DNA. Initiation of minicircle leading-strand synthesis involves the synthesis of an RNA primer which is removed in the last stage of replication. We report here the purification to near homogeneity of a structure-specific DNA endo-nuclease based on the RNase H activity of the enzyme on a poly(rA).poly(dT) substrate. RNase H activity gel analysis of whole cell and kinetoplast extracts shows that the enzyme is enriched in kinetoplast fractions. The DNA endonuclease activity of the enzyme is specific for DNA primers annealed to a template strand and requires an unannealed 5' tail. The enzyme cleaves 3' of the first base paired nucleotide releasing the intact tail. The purified enzyme migrates as a 32 kDa protein on SDS gels and has a Stoke's radius of 21.5 A and a sedimentation coefficient of 3.7 s, indicating that the protein is a monomer in solution with a native molecular mass of 32.4 kDa. These results suggest that the enzyme may be involved in RNA primer removal during minicircle replication.
Insights
Researchers purified a novel DNA endonuclease from Crithidia fasciculata, likely involved in removing RNA primers during mitochondrial DNA (kinetoplast DNA) replication. This enzyme
Area of Science:
- Molecular Biology
- Biochemistry
- Parasitology
Background:
- The mitochondrial DNA (kinetoplast DNA) of Crithidia fasciculata exists as a network of interlocked minicircles and maxicircles.
- Replication of minicircles involves unidirectional synthesis initiated by an RNA primer, which must be removed during the final stages.
Purpose of the Study:
- To purify and characterize a DNA endonuclease with RNase H activity from Crithidia fasciculata.
- To investigate the enzyme's potential role in RNA primer removal during minicircle replication.
Main Methods:
- Purification of a structure-specific DNA endonuclease using RNase H activity on a poly(rA).poly(dT) substrate.
- RNase H activity gel analysis of whole cell and kinetoplast extracts.
- Characterization of the enzyme's DNA endonuclease activity specificity and cleavage mechanism.
- Biophysical characterization including SDS-PAGE, Stoke's radius, and sedimentation coefficient.
Main Results:
- A DNA endonuclease with RNase H activity was purified to near homogeneity.
- The enzyme is enriched in kinetoplast fractions, suggesting a role in mitochondrial DNA metabolism.
- The endonuclease specifically cleaves DNA primers annealed to a template strand, requiring an unannealed 5' tail.
- The purified enzyme is a 32 kDa monomer, with a native molecular mass of 32.4 kDa.
Conclusions:
- The purified enzyme exhibits properties consistent with a role in RNA primer removal during Crithidia fasciculata minicircle replication.
- This enzyme represents a key factor in the DNA replication machinery of trypanosomatids.
- Further studies are warranted to confirm its precise function in vivo.