Rotations of the 2B Sub-domain of E. coli UvrD Helicase/Translocase Coupled to Nucleotide and DNA Binding

Abstract

E. coli UvrD is a superfamily 1 (SF1) DNA helicase and single stranded (ss) DNA translocase that functions in DNA repair, plasmid replication and as an anti-recombinase by removing RecA protein from ssDNA. UvrD couples ATP binding and hydrolysis to unwind double-stranded DNA (dsDNA) and translocate along ssDNA with 3′ to 5′ directionality. Although a UvrD monomer is able to translocate along ssDNA rapidly and processively, DNA helicase activity in vitro requires a minimum of a UvrD dimer. Previous crystal structures of UvrD bound to a ss-duplex DNA junction show that its 2B sub-domain exists in a “closed” state and interacts with the duplex DNA. Here we report a crystal structure of an apo form of UvrD in which the 2B sub-domain is in an “open” state that differs by a ~160° rotation of the 2B sub-domain. To study the rotational conformational states of the 2B sub-domain in various ligation states, a series of double cysteine UvrD mutants were constructed and labeled with fluorophores such that rotation of the 2B sub-domain results in changes in fluorescence resonance energy transfer (FRET). These studies show that the open and closed forms can interconvert in solution with low salt favoring the closed conformation and high salt favoring the open conformation in the absence of DNA. Binding of UvrD to DNA as well as ATP binding and hydrolysis also affect the rotational conformational state of the 2B sub-domain suggesting that 2B sub-domain rotation is coupled to the function of this nucleic acid motor enzyme.

Large Domain Movements Upon UvrD Dimerization and Helicase Activation

“…The dimer unwinds dsDNA with rates of ∼70 base pair (bp) s −1 (20,21,24,27). Rep and PcrA monomers also show no helicase activity and require oligomerization or an accessory protein for helicase activity in vitro (28-31).UvrD, Rep, and PcrA all contain four subdomains (1A, 2A, 1B, and 2B) (2,(32)(33)(34)(35), and the 2B subdomain can undergo a substantial rotation about a hinge region connecting it to the 2A subdomain (2,22,(32)(33)(34)(35)(36)(37). A structure of an apo UvrD monomer shows the 2B subdomain in an open conformation (35), whereas a structure of a UvrD monomer bound to an ss/ds DNA junction shows the 2B subdomain in a closed conformation (Fig.…”

“…These studies also showed that a UvrD dimer unwinds DNA with higher processivity than a monomer (22), but the basis for the higher processivity of the dimer remains unknown. One proposal is that dimerization activates helicase activity by influencing the rotational conformational state of the 2B subdomain (1,22,35,38,39).These studies point to the importance of understanding the dynamics of the 2B subdomain and its effect on the enzyme activities of SF1A helicases/translocases. Here we use single-molecule Significance UvrD helicase plays essential roles in multiple DNA metabolic processes.…”

“…A structure of an apo UvrD monomer shows the 2B subdomain in an open conformation (35), whereas a structure of a UvrD monomer bound to an ss/ds DNA junction shows the 2B subdomain in a closed conformation (Fig. 1A) in which the 2B domain contacts the duplex DNA (2).…”

“…The positions of the 2B subdomain in its open and closed structures differ by an ∼160°rotation (2,35). The rotational state of the 2B subdomain in apo UvrD is influenced by salt concentration and type, with a closed conformation favored at low salt and an open conformation favored at high salt (35).…”

“…These studies also showed that a UvrD dimer unwinds DNA with higher processivity than a monomer (22), but the basis for the higher processivity of the dimer remains unknown. One proposal is that dimerization activates helicase activity by influencing the rotational conformational state of the 2B subdomain (1,22,35,38,39).…”