DNA Interactions Probed by Hydrogen-Deuterium Exchange (HDX) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Confirm External Binding Sites on the Minichromosomal Maintenance (MCM) Helicase

Abstract

The archaeal minichromosomal maintenance (MCM) helicase from Sulfolobus solfataricus (SsoMCM) is a model for understanding structural and mechanistic aspects of DNA unwinding. Although interactions of the encircled DNA strand within the central channel provide an accepted mode for translocation, interactions with the excluded strand on the exterior surface have mostly been ignored with regard to DNA unwinding. We have previously proposed an extension of the traditional steric exclusion model of unwinding to also include significant contributions with the excluded strand during unwinding, termed steric exclusion and wrapping (SEW). The SEW model hypothesizes that the displaced single strand tracks along paths on the exterior surface of hexameric helicases to protect singlestranded DNA (ssDNA) and stabilize the complex in a forward unwinding mode. Using hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance MS, we have probed the binding sites for ssDNA, using multiple substrates targeting both the encircled and excluded strand interactions. In each experiment, we have obtained >98.7% sequence coverage of SsoMCM from >650 peptides (5-30 residues in length) and are able to identify interacting residues on both the interior and exterior of SsoMCM. Based on identified contacts, positively charged residues within the external waist region were mutated and shown to generally lower DNA unwinding without negatively affecting the ATP hydrolysis. The combined data globally identify binding sites for ssDNA during SsoMCM unwinding as well as validating the importance of the SEW model for hexameric helicase unwinding.DNA unwinding by hexameric replicative helicases is required for DNA replication elongation, providing the singlestranded DNA (ssDNA) 5 templates for leading and lagging strand synthesis. These hexameric helicase complexes form ring-like structures that preferentially encircle one of the DNA strands, providing stability in DNA binding and enhancing processivity for unwinding long stretches of DNA. In the steric exclusion model for unwinding, the motor domains within the central channel translocate along the encircled ssDNA while physically excluding the complementary ssDNA on the exterior. Although DNA unwinding by helicases can be effectively measured in gel-based and fluorescence assays, the specific molecular interactions with DNA are poorly described. Previously, we have proposed that the excluded strand is not a passive component of unwinding; rather, it will interact along specific exterior paths on the hexameric helicase surface to both stabilize the complex and promote forward unwinding (1). Although structures of hexameric helicases that include small stretches of ssDNA have been solved recently (2, 3), the interactions are constrained to central channel residues, and no molecular information is available at the duplex region or with respect to the excluded strand.Hexameric DNA replication helicases are known to exist across the three domains of life, including viruses. Althou...

Mechanisms and regulation of DNA replication initiation in eukaryotes

“…However, emerging lines of evidence are starting to suggest that the lagging strand may wrap around the external surface of the CMG and support helicase activity (Figure 7D). Consistent with this notion, DNA interactions with the external surface of archaeal MCM is integral to helicase function (Rothenberg et al, 2007, Costa et al, 2008, Graham et al, 2011, Graham et al, 2016). DNA binding to the MCM external surface appears at least partially facilitated by the NTD-A region, which can undergo large structural rearrangements to expose a DNA-binding site (Fletcher et al, 2003, Chen et al, 2005, Costa et al, 2008, Miller et al, 2014).…”

Bacterial DnaB helicase interacts with the excluded strand to regulate unwinding

“…Previously, we have shown that interactions with the excluded strand exist for the 3Ј-5Ј hexameric SsoMCM helicase (11,22). Using a similar smFRET approach, we sought to examine whether analogous contacts on the exterior of EcDnaB also interact with the excluded strand despite the opposing 5Ј-3Ј translocation polarity.…”

“…Mutating positively charged residues involved in the excluded strand interaction inhibited SsoMCM's unwinding activity (11,22). Gel-based fluorescent DNA unwinding assays were performed to determine whether these EcDnaB SEW mutants have any effect on activity.…”

MS-Based Approaches Enable the Structural Characterization of Transcription Factor/DNA Response Element Complex

“…In HDX experiments, the exchange rate of backbone amide hydrogens, which is affected by solvent accessibility and possible involvement in hydrogen bonding, can be directly determined by MS analysis. This technique has been broadly employed to study conformational changes [19,20], protein folding [21] and protein-protein interactions [22,23], as well as nucleic acid-protein complexes [24,25,26,27,28,29]. Among the MS3D techniques, chemical and photo-activated cross-linking (XL) are employed to generate stable covalent bridges between contiguous functional groups, which can reveal their mutual placement in the targeted assembly [13,16].…”