The T4 Phage SF1B Helicase Dda Is Structurally Optimized to Perform DNA Strand Separation

He, Xiaoping; Byrd, Alicia K.; Yun, Mi‐Kyung; Pemble, Charles W.; Harrison, David K.; Yeruva, Laxmi; Dahl, Christopher; Kreuzer, Kenneth N.; Raney, Kevin D.; White, Stephen W.

doi:10.1016/j.str.2012.04.013

Cited by 39 publications

(67 citation statements)

References 59 publications

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“…A recent report on Pif1 demonstrated that the contact site-size for Pif1 is ϳ6 -8 nt (20). Similarly the crystal structure of a related SF1B helicase, Dda, illustrated a similar contact site-size (17). Therefore, we assume that one molecule of Pif1 sequesters about 7 nucleotides when bound to ssDNA.…”

Section: An Increase In Overhang Length Results In Increased Product supporting

confidence: 63%

“…The rate of translocation (89 Ϯ 3 nt/s) is very similar to the rate of unwinding (75 Ϯ 12 bp/s). These results indicate that Pif1 unwinds dsDNA by an active mechanism similar to the T4 Dda helicase, which also belongs to the Pif1 subfamily of helicases and is also a highly active helicase even though Dda unwinds and translocates at much higher speeds (about 250 bp or nt/s) (17,37). Processivity for Pif1 translocation was 0.96, indicating that it translocates an average length of ϳ25 nt before dissociation.…”

Section: Tablementioning

confidence: 88%

“…Translocation on ssDNA-Translocation experiments were performed as described previously (17). All concentrations are after mixing.…”

Section: Methodsmentioning

confidence: 99%

“…The Pif1-like family includes yeast Pif1 and Rrm3, human Pif1 and HelB, bacteriophage T4 Dda, and Escherichia coli RecD and TraI. Crystal structures of RecD2 and Dda have revealed that the core motor comprises paired RecA-like domains and an SH3 domain (17,18). Domain 1B, which forms the pin structure, is proposed to split the incoming duplex.…”

mentioning

confidence: 99%

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Yeast Pif1 Helicase Exhibits a One-base-pair Stepping Mechanism for Unwinding Duplex DNA

Ramanagoudr-Bhojappa

Chib

Byrd

et al. 2013

Journal of Biological Chemistry

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Section: An Increase In Overhang Length Results In Increased Product supporting

confidence: 63%

Section: Tablementioning

confidence: 88%

“…Translocation on ssDNA-Translocation experiments were performed as described previously (17). All concentrations are after mixing.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Yeast Pif1 Helicase Exhibits a One-base-pair Stepping Mechanism for Unwinding Duplex DNA

Ramanagoudr-Bhojappa

Chib

Byrd

et al. 2013

Journal of Biological Chemistry

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“…Furthermore, SF1 helicases comprise the largest class and can be further subdivided into SF1A and SF1B, based on the direction of helicase translocation along the strand; SF1A helicases progress 3= to 5= and SF1B 5= to 3= (3). Despite their importance, SF1B helicases are still poorly characterized in comparison to SF1A and SF2 helicases, though some recent studies have expanded our knowledge (19)(20)(21). SF1B helicases are critical to a diverse range of processes, including DNA repair by human DNA helicase B (22), telomere regulation by Pif1 (23), and conjugative plasmid transfer by F TraI (24).…”

mentioning

confidence: 99%

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

2014

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The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.H elicases are molecular motors that drive the separation of duplex nucleic acid strands through the coupling of ATP hydrolysis to unwinding (1-3). As a result, helicases are key players in a wide variety of DNA and RNA metabolic processes, such as recombination, chromatin remodeling, and DNA transport (4-6). Most recently, helicases have emerged as potential therapeutic targets for diseases ranging from cancer to Gram-positive bacterial infections (7-18). Classified into six superfamilies, superfamily 1 and 2 (SF1 and SF2) helicases are the most similar, sharing up to seven conserved sequence motifs and core RecA-like folds (1-3). Furthermore, SF1 helicases comprise the largest class and can be further subdivided into SF1A and SF1B, based on the direction of helicase translocation along the strand; SF1A helicases progress 3= to 5= and SF1B 5= to 3= (3). Despite their importance, SF1B helicases are still poorly characterized in comparison to SF1A and SF2 helicases, though some recent studies have expanded our knowledge (19-21). SF1B helicases are critical to a diverse range of processes, including DNA repair by human DNA helicase B (22), telomere regulation by Pif1 (23), and conjugative plasmid transfer by F TraI (24).Conjugative plasmid transfer (CPT) mediates the propagation of antibiotic resistance genes and virulence factors within commensal and pathogenic bacteria (25-27) and relies on the helicase domain of a bifunctional protein to facilitate plasmid DNA transfer (24,(28)(29)(30). Early work characteri...

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Grip it and rip it: Structural mechanisms of DNA helicase substrate binding and unwinding

Bhattacharyya

Keck

2014

Protein Science

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Maintenance and faithful transmission of genomic information depends on the efficient execution of numerous DNA replication, recombination, and repair pathways. Many of the enzymes that catalyze steps within these pathways require access to sequence information that is buried in the interior of the DNA double helix, which makes DNA unwinding an essential cellular reaction. The unwinding process is mediated by specialized molecular motors called DNA helicases that couple the chemical energy derived from nucleoside triphosphate hydrolysis to the otherwise nonspontaneous unwinding reaction. An impressive number of high-resolution helicase structures are now available that, together with equally important mechanistic studies, have begun to define the features that allow this class of enzymes to function as molecular motors. In this review, we explore the structural features within DNA helicases that are used to bind and unwind DNA. We focus in particular on "aromatic-rich loops" that allow some helicases to couple single-stranded DNA binding to ATP hydrolysis and "wedge/pin" elements that provide mechanical tools for DNA strand separation when connected to translocating motor domains.

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The T4 Phage SF1B Helicase Dda Is Structurally Optimized to Perform DNA Strand Separation

Cited by 39 publications

References 59 publications

Yeast Pif1 Helicase Exhibits a One-base-pair Stepping Mechanism for Unwinding Duplex DNA

Yeast Pif1 Helicase Exhibits a One-base-pair Stepping Mechanism for Unwinding Duplex DNA

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

Grip it and rip it: Structural mechanisms of DNA helicase substrate binding and unwinding

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