Distinctive effects of domain deletions on the manganese-dependent DNA polymerase and DNA phosphorylase activities of Mycobacterium smegmatis polynucleotide phosphorylase.

Polynucleotide phosphorylase (PNPase) is a versatile enzyme that plays synthetic and degradative roles in bacterial RNA metabolism.1 In its degradative mode, PNPase catalyzes processive phosphorolysis of the 3′-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate (NDP) products. It its polymerase mode, PNPase uses NDPs as substrates to add NMPs to the 3′-OH terminus of RNA chains while expelling inorganic phosphate (Pi). The polymerase reaction is the microscopic reversal of the phosphorolysis reaction and both require a divalent cation. Insights to the structural organization and catalytic mechanism of PNPase have emerged from crystal structures of exemplary bacterial and eukaryal PNPases.2-6 PNPases are ring-shaped homotrimers with a central channel that admits RNA and within which the active site is located. The channel accommodates single-stranded RNA but excludes duplex RNA. Each protomer is composed of five domain modules (Fig. 1A). Two RNase PH-like domains comprise the core of the trimeric ring. The metal-binding site is located within the distal PH domain.4 An α-helical module separating the PH domains is disposed on the inferior surface of the ring. The C-terminal KH and S1 domains are on the opposite face of the ring and are conformationally mobile. In the Caulobacter PNPase structure, the three KH modules of the trimer form a narrow aperture that contacts the RNA and through which the RNA chain is threaded en route to the central cavity.6 Figure 1 Domain organization, purification, and RNA phosphorylase activity of mycobacterial PNPase Several studies have addressed the contributions of the KH and S1 domains to the activity of PNPase by deleting these modules, singly or in combination.3,5,7,8 In one report, deletion of the S1 domain of E. coli PNPase reduced the RNA phosphorylase specific activity by a factor of 50 and elicited a similar decrement in RNA binding affinity.8 In another study of the E. coli protein, deleting both the S1 and KH domains reduced RNA binding affinity by a factor of 8.3 By contrast, deleting the S1 domain of human PNPase did not affect RNA binding affinity or RNA phosphorylase activity, although mutations in the KH domain exerted severe effects.5 The disparate effects of C-terminal domain deletions on PNPase activity may reflect the use of different reaction conditions of varying stringency8 and/or variable effects of the domains deletions on the stability or conformation of the trimeric PNPase ring.3 Nonetheless, the emerging theme is that the KH and S1 domains are relevant for RNA binding at a distance from the active site. Although PNPase is generally regarding as an RNA modifying enzyme, exemplary bacterial PNPase enzymes are capable of synthesizing or degrading DNA in vitro when manganese or iron is provided in lieu of magnesium as the metal cofactor.9-13 Is this an in vitro quirk or might PNPase play a role in DNA metabolism? Recent genetic evidence implicates PNPase in bacterial DNA repair and mutagenesis in vivo.12-15 Moreover, Juan Alonso and colleagues make a good case for the direct involvement of Bacillus subtilis PNPase in DNA repair. They document that purified B. subtilis PNPase catalyzes Mn2+-dependent 3′ phosphorolysis of single-stranded (ss) DNA and Mn2+-dependent, template-independent polymerization of dADP at the 3′-OH terminus of a ssDNA primer.12,13 They provide genetic evidence (via studies of the effects of a ΔpnpA null allele on clastogen sensitivity and its epistasis relationships to null alleles of other DNA repair factors) that PNPase participates in the homologous recombination (HR) and nonhomologous end joining (NHEJ) pathways of B. subtilis double-strand break (DSB) repair in response to damage by hydrogen peroxide.12 And they show that ΔpnpA cells do not differ from wild-type cells with respect to the levels of RNA transcripts derived from genes involved in DNA repair and recombination.12 Although the mechanisms by which PNPase affects DNA repair are not yet clear, Alonso and colleagues suggest that PNPase reacts with broken DNA ends, either converting them from non-ligatable “dirty” breaks to clean ends that can be sealed by DNA ligase or by adding non-templated single-stranded 3′ tails that can then influence repair pathway choice.12,13 These studies of Bacillus PNPase prompted our interest in the DNA end modifying capacity of PNPase from mycobacteria, a genus of Actinobacteria that includes the human pathogen M. tuberculosis and its avirulent cousin M. smegmatis. Mycobacteria elaborate three genetically distinct pathways for the repair of DNA double-strand breaks: HR, NHEJ, and single-strand annealing.16 The M. smegmatis HR pathway comprises parallel RecA-dependent branches, one that relies on the AdnAB helicase-nuclease and a second that relies on RecO.16,17 (This is similar to the situation in B. subtilis, which has AddAB- and RecO-driven branches of RecA-dependent HR in vivo.18) The mycobacterial NHEJ system has been studied extensively in vivo and in vitro.19 The core components are the DNA end-binding protein Ku and DNA ligases D and C. Mycobacterial NHEJ at blunt and 5′-overhang DSBs is highly mutagenic, because nucleotides are added or subtracted from the broken ends prior to their sealing by ligase.20,21 At least some of these end-remodeling reactions are performed by LigD, which is a multifunctional enzyme composed of three autonomous catalytic modules – ligase (LIG), polymerase (POL) and 3′-phosphoesterase (PE) – fused into a single polypeptide.21-25 The polymerase and 3′-phosphoesterase activities of bacterial LigD are dependent on manganese26-28 and the ligase activities of LigD and LigC prefer manganese to magnesium.28 Here, we purified and characterize recombinant M. smegmatis PNPase and gauged the effects of active site mutations and domain deletions. As expected, we find that the full-length M. smegmatis PNPase is a homotrimeric enzyme with Mg2+·PO4-dependent RNA 3′-phosphorylase and Mg2+·ADP-dependent RNA polymerase activities. We show that the enzyme is a Mn2+·dADP-dependent template-independent DNA polymerase and a Mn2+·PO4-dependent DNA 3′-phosphorylase. Whereas serial subtraction of the S1 and KH domains incrementally suppresses the RNA-dependent activities of mycobacterial PNPase, the DNA polymerase and DNA phosphorylase activities are stimulated by deletion of the S1 domain. A PNPase that lacks the S1 and KH domains is as active in DNA synthesis and more active in DNA phosphorolysis than the full-length enzyme. These studies suggest that the S1 and KH domains act as RNA specificity filters for mycobacterial PNPase.