Multimeric BLM is dissociated upon ATP hydrolysis and functions as monomers in resolving DNA structures.

Bloom (BLM) syndrome is an autosomal recessive disorder characterized by an increased risk for many types of cancers. Previous studies have shown that BLM protein forms a hexameric ring structure, but its oligomeric form in DNA unwinding is still not well clarified. In this work, we have used dynamic light scattering and various stopped-flow assays to study the active form and kinetic mechanism of BLM in DNA unwinding. It was found that BLM multimers were dissociated upon ATP hydrolysis. Steady-state and single-turnover kinetic studies revealed that BLM helicase always unwound duplex DNA in the monomeric form under conditions of varying enzyme and ATP concentrations as well as 3'-ssDNA tail lengths, with no sign of oligomerization being discerned. Measurements of ATPase activity further indicated that BLM helicase might still function as monomers in resolving highly structured DNAs such as Holliday junctions and D-loops. These results shed new light on the underlying mechanism of BLM-mediated DNA unwinding and on the molecular and functional basis for the phenotype of heterozygous carriers of BLM syndrome.

Mutual inhibition of RecQ molecules in DNA unwinding.

Helicases make conformational changes and mechanical movements through hydrolysis of NTP to unwind duplex DNA (or RNA). Most helicases require a single-stranded overhang for loading onto the duplex DNA substrates. Some helicases have been observed to exhibit an enhanced unwinding efficiency with increasing length of the single-stranded DNA tail both by preventing reannealing of the unwound DNA and by compensating for premature dissociation of the leading monomers. Here we report a previously unknown mutual inhibition of neighboring monomers in DNA unwinding by the monomeric Escherichia coli RecQ helicase. With single molecule fluorescence resonance energy transfer microscopy, we observed that the unwinding initiation of RecQ at saturating concentrations was more delayed for a long rather than a short tailed DNA. In stopped-flow kinetic studies under both single and multiple turnover conditions, the unwinding efficiency decreased with increasing enzyme concentration for long tailed substrates. In addition, preincubation of RecQ and DNA in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate was observed to alleviate the inhibition. We propose that the mutual inhibition effect results from a forced closure of cleft between the two RecA-like domains of a leading monomer by a trailing one, hence the forward movements of both monomers are stalled by prohibition of ATP binding to the leading one. This effect represents direct evidence for the relative movements of the two RecA-like domains of RecQ in DNA unwinding. It may occur for all superfamily I and II helicases possessing two RecA-like domains.