Molecular Playground/PcrA Helicase

Helicases are nucleic acid–dependent ATP-ases that are capable of unwinding DNA or RNA duplex substrates. As a consequence, they play roles in almost every process in cells that involves nucleic acids, including DNA replication and repair, transcription, translation, ribosome synthesis (1). See: Space fill Model
(PDB entry 1pjr).

PcrA a Simple Model for Helicases

PcrA_Structure

PcrA is part of the replication machinery of the Geobacillus stearothermophilusa gram (+) bacteria, This helicase is part of the superfamily I of Helicases. Monomeric protein that is mainly alfa helical has the highly conserved Rec domians. This helicase was reported as a mutation in the gen PcrA from "Stapphylococcus aerous", this mutation was related to a deficiency in the replication of a reporter plasmid.[1]

PcrA Biochemistry

PcrA is has an ATPas activityt which directionality is from 3' to 5' helicase strand separation reaction. The enzyme shows a specificity for the DNA substrate in gel mobility assays with the preferred substrate being one containing both single and double stranded regions of DNA. In contrast to Rep and UvrD from E. coli, there is not evidence for dimerisation of the enzyme using gel filtration, or by crosslinking in the presence of combinations of Mg2+, nucleotides and DNA. Moreover, kcat for ATP hydrolysis is constant over a large range of protein concentrations. Therefore, the protein appears to be monomeric under all conditions tested, including in the structure of two crystal forms of PcrA.[2]

PcrA Helicase Mechanism: The Mexican Wave

Professor Dale B. Wigley' group in 1996-1999 was able to crystalize the intermediate states from PcrA, giving solution to the controversy of what kind of mechanism this helicase has. [3] Two crystal form of the enzyme, one couple with a 10 mer DNA and a non hydrolizable form of ATP (ATPnP)(3pjr), (Enzyme Subtrate Structure) and another a truncated form embebed in sulfate (2pjr) (Enzyme Product Structure), give a light in a model for how ATP hydrolysis results in motor movement along ssDNA. In the figure below step 1 (top) is the ATP free (product) ssDNA conformation. The DNA bases are labelled arbitrarily. On binding ATP, F626 creates a new binding pocket for base 6. Likewise, F64 destroys an acceptor pocket for base 2, forcing it to move to the position occupied by base 1. After ATP hydrolysis, the grip on base 6 is released. When the Y257 pocket is re-opened due to movement of F64, bases 3-6 can now flip through the acceptor pockets to their new positions. This model predicts that each ATP hydrolysis event will advance PcrA one base along ssDNA.[4]

Inchworm or Mexicanwave model

The Superfamily 1 (SF1)

PcrA share structural domains with the Rec helicases, like UvrD and RepD from E. coli, Superfamily 1 (SF1) helicases are probably the best characterized class, certainly from a structural perspective. All members characterized to date are bona fide helicases and α enzymes. Indeed, from their mode of translocation via the bases it is difficult to envisage how they could translocate along a duplex. However, they can have either A or B directional polarity.

• UvrD Structure (Helicase II/DNA complexed with sulfate 2is1)
• RecD Structure (REP helicase/DNA complex 1uaa)

About this Structure

1PJR is a Single protein structure of sequence from Geobacillus stearothermophilus. Full crystallographic information is available from OCA.

3D structures of helicase

Helicase

Additional Resources

For additional information, see: Bacterial Infections