Archaeal MCM has separable processivity, substrate choice and helicase domains

Abstract

The mini-chromosome maintenance (MCM) complex is the principal candidate for the replicative helicase of archaea and eukaryotes. Here, we describe a functional dissection of the roles of the three principal structural modules of the homomultimeric MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results include the first analysis of the central AAA+ domain in isolation. This domain possesses ATPase and helicase activity, defining this as the minimal helicase domain. Reconstitution experiments show that the helicase activity of the AAA+ domain can be stimulated by addition of the isolated N-terminal half in trans. Addition of the N-terminus influences both the processivity of the helicase and the choice of substrate that can be melted by the ATPase domain. The degenerate helix-turn-helix domain at the C-terminus of MCM exerts a negative effect on the helicase activity of the complex. These results provide the first evidence for extensive regulatory inter-domain communication within the MCM complex.

Figure 1.

(A) Demonstrates the domains contained within each of the eight constructs. (B) 2 µg of each protein were run on a 4–20% Tris-Glycine gel in MES buffer, before staining with Coomassie blue. (C) Shows the boundaries of each construct relative to the full-length protein and their molecular weights.

Figure 2.

(A) Figure 2A shows the UV 280 nm trace from gel filtration over a Superose 6 column using 25 µM of each of the indicated proteins. (B) Figure 2B shows the UV 280 nm trace from gel filtration over a Superdex 200 column using two concentrations for each protein. N-half: 200 µM, 25 µM. C-half 25 µM, 2.5 µM. AAA+ Core 25 µM, 2.5 µM. HTH 60 µM, 1 µM. The absorbance of each trace was normalised using the UNICORN program. (C) 25 µM Core domain, N-half or a mixture of both were incubated in the presence (+) or absence (−) of glutaraldehyde prior to SDS-PAGE on a 4–12% polyacrylamide gradient gel. (D) 25 µM AAA+ Core were run alone or mixed with 200 µM N-half on a Superdex 200 column. 80 µl samples were collected and electrophoresed on a 10% polyacrylamide gel. (E) His-tagged AAA+ domain was used in pull-down experiments with untagged N-terminal domain. 2–1% N-terminal domain input, 20–10% N-terminal domain input, Bead—Ni-NTA beads incubated with N-terminus, Beads + Core – Ni-NTA beads bound to AAA+ domain incubated with N-terminus.

Figure 3.

EMSA assays were performed as described (15), (A) Representative EMSA assay with 0, 50, 100, 150, 200, 250, 300, 350, 400, 500 and 600 nM wild-type MCM (B) Figure 3B demonstrates the percentage of DNA bound at the indicated concentration for constructs: full length, ▵A, ▵HTH and ▵A/HTH. (C) Figure 3C demonstrates the results for N-half, C-half, AAA+ core, HTH and an equimolar mix of N-half and AAA+ core. (D) EMSA assays employing 6 µM Core and/or N-half protein. The positions of free DNA and the relevant complexes are indicated.

Figure 4.

ATPase activity of the different constructs is shown relative to the activity of the full-length construct (wild-type activity of 1 µM MCM is 35 nM s⁻¹).

Figure 5.

(A) Determination of the polarity of Sso MCM helicase activity. 25, 50 or 100 ng of Sso MCM or 100 ng of Mth MCM were incubated with substrates designed to have 3′, 5 or 3′ and 5′ single–stranded extensions. The positions of substrate (S) and product (P) are indicated. (B) Figure 5B shows the helicase activity of the different deletion constructs at various concentrations, N-half and HTH are omitted as no activity was detected. (C) Figure 5C compares the activity of the AAA+ core with an equivalent construct possessing a K > A mutation in the Walker A motif.

Figure 6.

Indicated concentrations of the core AAA+ domain were incubated with substrates with (A) both 3′ and 5′ single-stranded extensions (B) 3′ single-stranded extensions. (C and D) In Figures 5C and D, indicated concentrations of wild-type full-length MCM (WT FL), wild-type core AAA+ domain (WT Core) or core AAA+ domain with a mutation in the Walker A motif (K346A Core) were incubated in the presence of substrates containing (C) a 5′ single-stranded extension, (D) no single-stranded extensions. (E) 2.5 μM wild-type full-length MCM (FL), 2.5 μM core AAA+ domain with a mutation in the Walker A motif (WA), or 0.25 μM and 0.5 μM wild-type core AAA+ domain were incubated with a double-stranded DNA substrate in the presence of 10 mM ADP, ADP-AlFl₄, AMP-PNP or ATP as indicated.

Figure 7.

(A) 600 nM core domain MCM was incubated with 3′-tailed substrate in the presence of the indicated concentrations of BSA or N-half. (B) 300 nM core domain was incubated with 3′-tailed substrate in the presence of the indicated concentrations of wild-type N-half (WT) or N-half with a mutation in the DNA-binding β-hairpin motif (KR246AA). (C) 300 nM core domain was incubated with 5′-tailed substrate in the presence of the indicated concentrations of wild-type N-half (WT) or N-half with a mutation in the DNA-binding β-hairpin motif (KR246AA). (D) 300 nM core domain was incubated with a double-stranded DNA substrate in the presence of the indicated concentrations of wild-type N-half (WT) or N-half with a mutation in the DNA binding β-hairpin motif (KR246AA). (E) The core domain was incubated with various length substrates in the presence of the indicated concentrations of wild-type N-half (WT) or N-half with a mutation in the DNA binding β-hairpin motif (KR246AA), and the ratio of long:short fragments unwound was calculated.

Figure 8.

Summary of interactions detected within the MCM complex. The dotted lines indicate that the N-half-core interactions could be occurring in cis and/or trans.