Characterization of the mycobacterial AdnAB DNA motor provides insights into the evolution of bacterial motor-nuclease machines Journal Article
| Authors: | Unciuleac, M. C.; Shuman, S. |
| Article Title: | Characterization of the mycobacterial AdnAB DNA motor provides insights into the evolution of bacterial motor-nuclease machines |
| Abstract: | Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (kcat 415 s-1), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3′→5′ translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the "leading" AdnB and "lagging" AdnA motor domains track in tandem, 3′ to 5′, along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5′ and 3′ terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5′ strand, while the AdnB nuclease cleaves the displaced 3′ strand. We present evidence that the distinctive "molecular ruler" function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5′-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Keywords: | controlled study; unclassified drug; genetics; nonhuman; binding affinity; protein function; metabolism; dna repair; genes; protein dna binding; protein binding; enzymology; wild type; bacteria (microorganisms); physiology; dna strand breakage; evolution, molecular; bacterial protein; chemistry; bacterial proteins; dna; double stranded dna; molecular evolution; recombinant proteins; recombinant protein; wild types; dna breaks, double-stranded; double stranded dna break; mutagenesis, site-directed; protein structure, tertiary; helicase; adenosine triphosphate; single stranded dna; dna, single-stranded; adenosine triphosphatase; site directed mutagenesis; bacterial dna; mycobacterium; adenosine triphosphatases; protein tertiary structure; streptavidin; dna helicases; dna cleavage; motor domain; mycobacterial; dna denaturation; mycobacterium smegmatis; molecular ruler; protein adnab; bacterial genetics; dna duplexes; exodeoxyribonuclease v; cofactors; dna double strand breaks; n-terminals; translocases; biochemical characterization; displacement assay; dna length; dna motors; heterodimers; processing machines; single strand dna (ss-dna); molecular motor; single stranded dna dependent atpase |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 285 |
| Issue: | 4 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2010-01-22 |
| Start Page: | 2632 |
| End Page: | 2641 |
| Language: | English |
| DOI: | 10.1074/jbc.M109.076133 |
| PUBMED: | 19920138 |
| PROVIDER: | scopus |
| PMCID: | PMC2807320 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 5" - "Export Date: 20 April 2011" - "CODEN: JBCHA" - "Source: Scopus" |
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