Distinctive effects of domain deletions on the manganese-dependent DNA polymerase and DNA phosphorylase activities of Mycobacterium smegmatis polynucleotide phosphorylase Journal Article
| Authors: | Unciuleac, M. C.; Shuman, S. |
| Article Title: | Distinctive effects of domain deletions on the manganese-dependent DNA polymerase and DNA phosphorylase activities of Mycobacterium smegmatis polynucleotide phosphorylase |
| Abstract: | Polynucleotide phosphorylase (PNPase) plays synthetic and degradative roles in bacterial RNA metabolism; it is also suggested to participate in bacterial DNA transactions. Here we characterize and compare the RNA and DNA modifying activities of Mycobacterium smegmatis PNPase. The full-length (763-aa) M. smegmatis PNPase is a homotrimeric enzyme with Mg2+•PO 4-dependent RNA 3′-phosphorylase and Mg2+•ADP- dependent RNA polymerase activities. We find that the enzyme is also a Mn 2+•dADP-dependent DNA polymerase and a Mn2+• PO4-dependent DNA 3′-phosphorylase. The Mn2+• DNA and Mg2+•RNA end modifying activities of mycobacterial PNPase are coordinately ablated by mutating the putative manganese ligand Asp526, signifying that both metals likely bind to the same site on PNPase. Deletions of the C-terminal S1 and KH domains of mycobacterial PNPase exert opposite effects on the RNA and DNA modifying activities. Subtracting the S1 domain diminishes RNA phosphorylase and polymerase activity; simultaneous deletion of the S1 and KH domains further cripples the enzyme with respect to RNA substrates. By contrast, the S1 and KH domain deletions enhance the DNA polymerase and phosphorylase activity of mycobacterial PNPase. We observe two distinct modes of nucleic acid binding by mycobacterial PNPase: (i) metal-independent RNA-specific binding via the S1 domain, and (ii) metal-dependent binding to RNA or DNA that is optimal when the S1 domain is deleted. These findings add a new dimension to our understanding of PNPase specificity, whereby the C-terminal modules serve a dual purpose: (i) to help capture an RNA polynucleotide substrate for processive 3′ end additions or resections, and (ii) to provide a specificity filter that selects against a DNA polynucleotide substrate. © 2013 American Chemical Society. |
| Keywords: | gene deletion; dna polymerase; protein dna binding; enzyme activity; phosphorylation; bacteria (microorganisms); genetic vectors; rna; dna; crystal structure; models, molecular; enzyme specificity; enzymes; substrates; electrophoresis, polyacrylamide gel; expression vector; magnesium ion; mycobacterium smegmatis; rna polymerase; protein rna binding; dna-directed dna polymerase; nucleotides; polymerase activities; polymers; bacteria; nucleic acid binding protein; manganese; polynucleotides; rna metabolisms; new dimensions; polynucleotide phosphorylase; polyribonucleotide nucleotidyltransferase; phosphorylases |
| Journal Title: | Biochemistry |
| Volume: | 52 |
| Issue: | 17 |
| ISSN: | 0006-2960 |
| Publisher: | American Chemical Society |
| Date Published: | 2013-04-30 |
| Start Page: | 2967 |
| End Page: | 2981 |
| Language: | English |
| DOI: | 10.1021/bi400281w |
| PROVIDER: | scopus |
| PUBMED: | 23560592 |
| PMCID: | PMC3689434 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 3 June 2013" - "CODEN: BICHA" - "Source: Scopus" |
Altmetric
Citation Impact
BMJ Impact Analytics
Related MSK Work