Abstract
BackgroundThe cancer-prone and accelerated aging disease Werner syndrome is caused by loss of function of the WRN gene product that possesses ATPase, 3' to 5' helicase and 3' to 5' exonuclease activities. Although WRN has been most prominently suggested to function in telomere maintenance, resolution of replication blockage and/or recombinational repair, its exact role in DNA metabolism remains unclear. WRN is the only human RecQ family member to possess both helicase and exonuclease activity, but the mechanistic relationship between these activities is unknown. In this study, model single-stranded and 3' overhang DNA substrates of varying length and structure were used to examine potential coordination between the ATPase/helicase and exonuclease activities of WRN.ResultsOur results show that WRN can not only bind to but also catalyze the 3' to 5' degradation of single-stranded and 3' overhang DNA substrates, structures that were previously thought to be refractory to WRN exonuclease activity. The length of the single-stranded regions in these structures is a critical parameter in determining both the binding affinity and the level of exonuclease activity of WRN. Most importantly, specific nucleotide cofactors dramatically stimulate WRN exonuclease activity on these substrates, with conditions that permit ATP hydrolysis not only resulting in enhanced exonuclease activity but also altering its length dependence on these structures. Parallel experiments show that a deletion mutant containing only the WRN exonuclease domain lacks both this DNA length and nucleotide cofactor dependence, demonstrating that the interaction of the ATPase/helicase domain of WRN with the DNA substrate has a profound influence on exonuclease activity.ConclusionOur results indicate that, under conditions that permit ATP hydrolysis, there is a dynamic and cooperative relationship between the distinct ATPase/helicase and exonuclease domains of WRN with regard to their orientation on DNA. Based on these results, models are proposed for the coordinated, unidirectional 3' to 5' movement of the helicase and exonuclease domains of WRN on DNA that should be informative for elucidating its function in genome maintenance.
Highlights
The cancer-prone and accelerated aging disease Werner syndrome is caused by loss of function of the WRN gene product that possesses ATPase, 3' to 5' helicase and 3' to 5' exonuclease activities
WRN exonuclease activity on single-stranded DNA is length dependent In previous studies, we have observed that WRN acts very efficiently on long DNA substrates (> 50 bp/nt) that contained at least partial single-stranded character [30,31]
Individual single-stranded oligomers ranging in lengths from 24 to 80 nt [G24, G30, G35, G40, G45 and G80, each sharing a minimum of 24 nt at their 3' ends] were incubated with WRN for 15 min without ATP and DNA products were separated by denaturing polyacrylamide (12%) gel electrophoresis (PAGE)
Summary
The cancer-prone and accelerated aging disease Werner syndrome is caused by loss of function of the WRN gene product that possesses ATPase, 3' to 5' helicase and 3' to 5' exonuclease activities. Individuals with WS normally die prior to age 50 from either heart disease or cancer This segmental progeroid condition appears to be due to increased genomic instability caused by the loss of function of a single gene product, WRN. A cellular deficiency in a RecQ family member results in an illegitimate recombination phenotype, suggesting key roles for RecQ helicases in minimizing largescale genome rearrangements Their exact metabolic functions remain unclear, commonly proposed roles for RecQ helicases involve participation in the disruption of aberrant recombination and in the resolution of replication blockage by fork regression or recombinational repair pathways [10,11,12]. Accumulation of senescent cells and/or loss of cells by apoptosis have been proposed as putative mechanisms at work in development of aging phenotypes in WS and during normal aging [18,19]
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