Currently, two types of substrates for prokaryotic RNase HII have been identified. The first type of RNase HII substrates are RNA NVP-BEZ235 PI3K inhibitor primers generated during DNA replication. Although the major pathway for LY294002 Okazaki primer removal in eukaryotes is independent of RNase H activity, these structures might also be removed through RNase HII. Using an in vitro model, it was demonstrated that RNase H type 2 proteins cleave RNA primers in Okazaki fragments, leaving the last ribonucleotide of the primer attached to the DNA, which is subsequently cleaved by FEN1. This mechanism was consistent with the observation that human RNase H2 localizes to replication foci. The involvement of RNase H type 2 in Okazaki fragment processing has also been confirmed in archaea. However, in Escherichia coli, the enzyme responsible for the removal of RNA primers during Okazaki fragment maturation is polymerase I, encoded by polA. Apart from PolI, RNase H proteins have also been implicated in RNA primer removal in bacteria. The second type of substrate for RNase HII are single ribonucleotides embedded within DNA duplex. Until recently, the incorporation of ribonucleotides other than Okazaki fragments within the DNA double helix has been a neglected biological phenomenon. However, several recent studies have shown that ribonucleotides are indeed present within DNA, and ribonucleotide triphosphates incorporation is evolutionarily conserved from bacteria to eukaryotes. In fact, ribonucleotides might be the most common lesion in the DNA double helix. For example, the replicative polymerases ��, ��, and �� in S. cerevisiae have been shown to insert one ribonucleotide for every 625, 5000 and 1250 deoxyribonucleotide triphosphates, respectively. This activity results in the incorporation of approximately 10 thousand ribonucleotides per round of replication. In yeast, the removal of ribonucleotides embedded within the DNA double helix primarily occurs through RNase H2 and to some extent, RNase H1 and topoisomerase I. Although genetic analyses in yeast have excluded the involvement of nucleotide excision repair, the minor involvement of base excision repair could not be ruled out. Intriguingly, contrasting observations have been made in E. coli where, apart from RNase HII activity, the removal of ribonucleotide monophosphate primarily involved nucleotide excision repair, while the involvement of base excision repair and mismatch repair was minimal. Both, increased RNase HII substrates and RNase HII deficiency have been associated with genome instability in several organisms. Thus, the aim of the present study was to investigate whether rnhB, presumably encoding RNase HII, influences RNase HII substrate levels and the genome stability in the M. tuberculosis model organism M. smegmatis. M. smegmatis genome contains two putative RNases H- one RNase H class I enzyme encoded by rnhA gene one RNase H class II enzyme encoded by rnhB gene.