Structure and primase-mediated activation of a bacterial dodecameric replicative helicase

Abstract

Replicative helicases are essential ATPases that unwind DNA to initiate chromosomal replication. While bacterial replicative DnaB helicases are hexameric, Helicobacter pylori DnaB (HpDnaB) was found to form double hexamers, similar to some archaeal and eukaryotic replicative helicases. Here we present a structural and functional analysis of HpDnaB protein during primosome formation. The crystal structure of the HpDnaB at 6.7 Å resolution reveals a dodecameric organization consisting of two hexamers assembled via their N-terminal rings in a stack-twisted mode. Using fluorescence anisotropy we show that HpDnaB dodecamer interacts with single-stranded DNA in the presence of ATP but has a low DNA unwinding activity. Multi-angle light scattering and small angle X-ray scattering demonstrate that interaction with the DnaG primase helicase-binding domain dissociates the helicase dodecamer into single ringed primosomes. Functional assays on the proteins and associated complexes indicate that these single ringed primosomes are the most active form of the helicase for ATP hydrolysis, DNA binding and unwinding. These findings shed light onto an activation mechanism of HpDnaB by the primase that might be relevant in other bacteria and possibly other organisms exploiting dodecameric helicases for DNA replication.

Figure 1.

Crystal structure of HpDnaB dodecamer. (A) Schematic representation of HpDnaB domain organization. (B) Surface representation of His-HpDnaB crystal structure with one hexamer colored according to the domain organization described in A) and the second one in green. The second CTD-ring, absent in the crystal structure has been modeled and is shown as a cartoon. (C) Top panels, structures of NTD-rings of His-HpDnaB, apo BstDnaB and GP40. The inset depicts a schematic representation of the apparent six-fold symmetry of His-HpDnaB NTD-rings. Bottom panels, surface representation of HpDnaB, apo BstDnaB and GP40 CTD-ring structures. Subunits are colored in alternating shades for clarity, linker helices are colored in yellow, HPI helices α15 (orange) and α16 (magenta) are shown as cylinders.

Figure 2.

Conformation of the dodecamer and site-directed mutagenesis study of the hexamer–hexamer interface. (A) Close-up view of the hexamer–hexamer interface with side chains of participating residues shown as sticks. Helices are labeled and the histidine-tag is colored in magenta. (B) Experimental SAXS curves of His-HpDnaB (blue) and HpDnaB (orange). (C) Representative reference-free 2D class averages of His-HpDnaB and HpDnaB. (D) Size exclusion chromatograms (280 nm) of HpDnaB and mutants L4A, Q8A, E80A, and Δ9 performed on a Superdex 200 increase (GE, 0.45 ml.min⁻¹). MALS weight-averaged molar masses are indicated as dotted lines.

Figure 3.

Effects of ATP and AMPPNP on the HpDnaB interaction with DNA. (A) Fluorescence anisotropy measurements of HpDnaB binding to either 5′- FAM labeled 20mer ssDNA (20dT) or dsDNA in the presence and absence of nucleotide (ATP and AMPPNP). The curves represent the mean of three independent experiments. (B) Similar experiments performed with 5′- FAM labeled 50mer ssDNA (50dT). (C) Size exclusion chromatograms (280 nm) of HpDnaB alone (light blue) or mixed with a 20dT oligonucleotide (75 μM) and 5 mM ATP (dark blue). MALS weight-averaged molar masses are indicated as dotted lines. The SDS-PAGE analysis shows the HpDnaB protein elution fractions. (D) SEC-MALS experiment performed as in C) except that HpDnaB was incubated with 5 mM AMPPNP and the running buffer contained 0.5 mM AMPPNP. Three samples were analyzed: HpDnaB (blue) and HpDnaB with 45 μM (orange) or 75 μM (red) 20dT. The SDS-PAGE analysis shows that HpDnaB elutes slightly later in the presence of ssDNA and AMPPNP.

Figure 4.

Interaction of HpDnaG^HBD dissociates HpDnaB dodecamer. (A) Size-exclusion chromatograms (280 nm) of HpDnaB (light blue), HpDnaG^HBD (orange) or the reconstituted primosome HpDnaB + HpDnaG^HBD (green). MALS weight-averaged molar masses measurements are indicated as dotted lines. The SDS-PAGE analysis shows the proteins contained in the elution fractions. (B) Experimental scattering curve of the HpDnaB•HpDnaG^HBD peak (grey) compared to theoretical curves of HpDnaB dodecamer (orange), and model of the HpDnaB₆•HpDnaG^HBD₃ (green). The inset shows the improved fit obtained from a MES containing a mixture of HpDnaB₆•HpDnaG^HBD₃ and HpDnaB dodecamer models.

Figure 5.

HpDnaG^HBD primes HpDnaB activities. (A) Single-stranded DNA binding activity of HpDnaB and HpDnaB•HpDnaG^HBD complex in the presence of ATP (0.5 mM) or AMPPNP (0.5 mM) as measured by fluorescence anisotropy. (B) Unwinding of a fluorophore (F)/quencher (Q)-labeled forked DNA substrate (shown schematically in inset) by HpDnaB (black) or HpDnaB•HpDnaG^HBD (green). The curve represents the mean of three independent experiments and the standard deviations are indicated for each measurement as error bars. (C) Schematic illustration of the proposed model of HpDnaG^HBD activation of the double hexamer on ssDNA during replication initiation in Helicobacter pylori.