Jensen Martin Borch, Dunn Christopher A, Keijzers Guido, Kulikowicz Tomasz, Rasmussen Lene Juel, Croteau Deborah L, Bohr Vilhelm A
Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark.
Aging (Albany NY). 2012 Nov;4(11):790-802. doi: 10.18632/aging.100506.
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.
RECQL4是人类RecQ解旋酶家族的五个成员之一，与三种表现出加速衰老、发育异常和癌症易感性的综合征有关。在本研究中，我们纯化了携带先前报道的患者突变的三种RECQL4变体。对这三种突变蛋白进行了RECQL4已知生化活性的分析：DNA结合、双链DNA解旋、ATP水解和单链DNA退火。此外，还评估了突变蛋白的稳定性以及对激光诱导的DNA损伤位点的募集情况。一种突变体解旋酶失活，ATP酶活性微弱，可能在结构上受到损害，而另外两种突变体的解旋酶和ATP酶活性则大大降低。对于任何一种突变体，其余的生化活性和募集到损伤位点的能力均未受到明显损害。我们的研究结果证明了一种一致的功能缺陷模式，并进一步支持了RECQL4除了在复制起始中依赖其N端的作用外，还具有依赖解旋酶的细胞功能，这一功能可能是RECQL4相关疾病表型的基础。
