Abstract
RECQL4 is one of five members of the human RecQ helicase family, and is implicated in three syndromes displaying accelerating aging, developmental abnormalities and a predisposition to cancer. In this study, we purified three variants of RECQL4 carrying previously reported patient mutations. These three mutant proteins were analyzed for the known biochemical activities of RECQL4: DNA binding, unwinding of duplex DNA, ATP hydrolysis and annealing of simplex DNA. Further, the mutant proteins were evaluated for stability and recruitment to sites of laser-induced DNA damage. One mutant was helicase-dead, had marginal ATPase activity and may be structurally compromised, while the other two showed greatly reduced helicase and ATPase activities. The remaining biochemical activities and ability to recruit to damage sites were not significantly impaired for any of the mutants. Our findings demonstrate a consistent pattern of functional deficiency and provide further support for a helicase-dependent cellular function of RECQL4 in addition to its N-terminus-dependent role in initiation of replication, a function that may underlie the phenotype of RECQL4-linked disease.
Highlights
The RecQ family of helicases is conserved across multiple species and has been firmly linked to genomic maintenance [1,2,3]
Our data shows that the helicase and ATPase activities are strongly affected for all three mutants, similar to what was recently found for the c.1390+2delT mutation found in other RAPADILINO patients that causes a 44 amino acid deletion just prior to the helicase domain [46]
In view of the ATP-dependent nature of the helicase activity, this could be interpreted in two ways: either a given mutation prevents ATP hydrolysis, which in turn inactivates the helicase function, or the mutation disrupts helicase activity, which results in ATP not being hydrolyzed regardless of the capacity for doing so
Summary
The RecQ family of helicases is conserved across multiple species and has been firmly linked to genomic maintenance [1,2,3]. Five RecQ helicases are present in humans: RECQL1, Bloom (BLM), Werner (WRN), RECQL4 and RECQL5. A central helicase domain, which allows for 3’ to 5’ unwinding of DNA, is conserved across the family [1,4]. Of the five human RecQ helicases, three (WRN, BLM and RECQL4) are associated with diseases involving segmental premature aging and cancer predisposition [8,9,10,11]. While the mechanisms behind Werner (OMIM 277700) and Bloom Syndromes (OMIM 210900) are not yet fully understood, the responsible proteins are at this stage relatively welldescribed as being required to resolve DNA secondary structures, and prevent inadvertent homologous recombination, respectively [12,13,14,15,16,17,18,19]. The biological role of RECQL4 is not nearly as well understood [5,20,21,22,23,24,25]
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