RNA primer removal and gap filling on a model minicircle replication intermediate

Abstract

Replication of kinetoplast DNA minicircles in Crithidia fasciculata occurs by a unidirectional mechanism involving continuous synthesis of one strand (L strand) and discontinuous synthesis of the complementary strand (H strand). L-strands are initiated by RNA priming at alternate origins (A and B) resulting in daughter molecules with a single nick or gap in the L strand at either ori A or ori B. Some of the gapped molecules contain ribonucleotides at the 5′ side of the gap. We have investigated the ability of recombinant forms of kinetoplast replication proteins, DNA polymerase β and structure specific endonuclease 1, to repair gaps in a model minicircle substrate. Structure specific endonuclease 1 was shown to efficiently remove all ribonucleotides from the 5′ side of the model substrate by stepwise cleavage of the RNA primer. Polymerase β was then able to extend the 3′ terminus of the gap to yield a nicked molecule capable of covalent joining by a DNA ligase. These results demonstrate that the nuclease and polymerase enzymes present at antipodal protein complexes flanking the kinetoplast disk are capable of complete RNA primer removal and subsequent gap filling of newly synthesized minicircle L strands.