ABSTRACT
We characterize Mycobacterium smegmatis FenA as a manganese-dependent 5′-flap endonuclease homologous to the 5′-exonuclease of DNA polymerase I. FenA incises a nicked 5′ flap between the first and second nucleotides of the duplex segment to yield a 1-nucleotide gapped DNA, which is then further resected in dinucleotide steps. Initial FenA cleavage at a Y-flap or nick occurs between the first and second nucleotides of the duplex. However, when the template 3′ single strand is eliminated to create a 5′-tailed duplex, FenA incision shifts to between the second and third nucleotides. A double-flap substrate with a mobile junction (mimicking limited strand displacement synthesis during gap repair) is preferentially incised as the 1-nucleotide 3′-flap isomer, with the scissile phosphodiester shifted by one nucleotide versus a static double flap. FenA efficiently removes the 5′ App(dN) terminus of an aborted nick ligation reaction intermediate, thereby highlighting FenA as an agent of repair of such lesions, which are formed under a variety of circumstances by bacterial NAD+-dependent DNA ligases and especially by mycobacterial DNA ligases D and C.
IMPORTANCE Structure-specific DNA endonucleases are implicated in bacterial DNA replication, repair, and recombination, yet there is scant knowledge of the roster and catalytic repertoire of such nucleases in Mycobacteria. This study identifies M. smegmatis FenA as a stand-alone endonuclease homologous to the 5′-exonuclease domain of mycobacterial DNA polymerase 1. FenA incises 5′ flaps, 5′ nicks, and 5′ App(dN) intermediates of aborted nick ligation. The isolated N-terminal domain of M. smegmatis Pol1 is also shown to be a flap endonuclease.
Distinctive Effects of Domain Deletions on the Manganese-Dependent DNA Polymerase and DNA Phosphorylase Activities of Mycobacterium smegmatis Polynucleotide Phosphorylase
