OverviewWerner syndrome protein (WRN) is one of a family of five human RecQ helicases implicated in the maintenance of genome stability. The conserved RecQ family also includes RecQ1, Bloom syndrome protein (BLM), RecQ4, and RecQ5 in humans (see additional reviews in this issue), as well as Sgs1 in Saccharomyces cerevisiae, Rqh1 in Schizosaccharomyces pombe, and homologs in Caenorhabditis elegans, Xenopus laevis, and Drosophila melanogaster [1]. Defects in three of the RecQ helicases, RecQ4, BLM, and WRN, cause human pathologies linked with cancer predisposition and premature aging [1][2][3][4][5]. Mutations in the WRN gene are the causative factor of Werner syndrome (WS). WRN is one of the best characterized of the RecQ helicases and is known to have roles in DNA replication and repair, transcription, and telomere maintenance [1][2][3][4][5][6]. Studies both in vitro and in vivo indicate that the roles of WRN in a variety of DNA processes are mediated by post-translational modifications, as well as several important protein-protein interactions [1,2,7]. Many of these functions of WRN in genome maintenance, as well as the clinical characteristics of WS, have been recently reviewed [8][9][10][11][12]. In this work, we will summarize some of the early studies on the cellular roles of WRN and highlight the recent findings that shed some light on the link between the protein and its cellular functions with the disease pathology. Molecular Genetics of Werner Syndrome (WS)WS is a rare autosomal recessive progeroid disorder characterized by the development of cataracts, changing of skin conditions, bird-like facies, atypical short stature, and premature graying or thinning of the hair [9]. Patients also often develop hypogonadism, osteoporosis, diabetes mellitus, artherosclerosis [13], and cancers, particularly sarcomas [14]. Onset of symptoms usually occurs in the third decade of life, and health subsequently declines with median age at death between 47-54 years [13]. Because WS presents with early-onset of conditions commonly seen in the aged, it is a good model system for the study of mechanisms of normal aging [15,16]. WRN BiochemistryThe causative factor of the majority of WS is mutation in the WRN gene, which codes for a member of the highly conserved RecQ family of helicases. While several different mutations within the gene are seen in WS, most result in production of truncated WRN protein [13]. 1Address correspondence to: Dr. Vilhelm A. Bohr, NIH Biomedical Research Center, 251 Bayview Boulevard, Baltimore, MD 21224; BohrV@grc.nia.nih.gov. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the...

Background: RECQL4 is a RecQ helicase mutated in Rothmund-Thomson Syndrome (RTS) and has a functional role in DNA replication and repair. Results: RECQL4-depleted and RTS patient cells show telomere abnormalities and that RECQL4 interacts with telomeric DNA and related proteins. Conclusion: RECQL4 is involved in telomere maintenance. Significance: The RecQ helicase RECQL4 is involved in telomere replication and maintenance. This establishes a connection between telomere function and a disease with premature aging phenotype.

Eukaryotic Okazaki fragment maturation requires complete removal of the initiating RNA primer before ligation occurs. Polymerase ␦ (Pol ␦) extends the upstream Okazaki fragment and displaces the 5-end of the downstream primer into a single nucleotide flap, which is removed by FEN1 nuclease cleavage. This process is repeated until all RNA is removed. However, a small fraction of flaps escapes cleavage and grows long enough to be coated with RPA and requires the consecutive action of the Dna2 and FEN1 nucleases for processing. Here we tested whether RPA inhibits FEN1 cleavage of long flaps as proposed. Surprisingly, we determined that RPA binding to long flaps made dynamically by polymerase ␦ only slightly inhibited FEN1 cleavage, apparently obviating the need for Dna2. Therefore, we asked whether other relevant proteins promote long flap cleavage via the Dna2 pathway. The Pif1 helicase, implicated in Okazaki maturation from genetic studies, improved flap displacement and increased RPA inhibition of long flap cleavage by FEN1. These results suggest that Pif1 accelerates long flap growth, allowing RPA to bind before FEN1 can act, thereby inhibiting FEN1 cleavage. Therefore, Pif1 directs long flaps toward the two-nuclease pathway, requiring Dna2 cleavage for primer removal.During eukaryotic DNA replication, the lagging strand is replicated via synthesis and maturation of Okazaki fragments. These fragments are short stretches of DNA that are joined to generate a continuous strand (1). Each fragment is initiated when DNA polymerase ␣/primase (Pol ␣) 3 makes an RNA/ DNA primer, synthesizing ϳ10 nucleotides (nt) of RNA followed by 10 -20 nt of DNA (2). The primer is then extended by a complex of DNA polymerase ␦ (Pol ␦), the sliding clamp, proliferating cell nuclear antigen (PCNA), and the clamp loader, replication factor C (RFC). When Pol ␦ encounters the 5Ј-end of the downstream Okazaki fragment, it displaces it into a flap. Cleavage of the flap by nucleases generates a nick, which is subsequently sealed by DNA ligase I to form continuous double-stranded DNA (3, 4).One pathway for cleavage of the flap employs flap endonuclease 1 (FEN1). FEN1 is a single-strand, structure-specific endonuclease that enters the 5Ј-end of the flap and tracks to the base for cleavage (3,5,6). Following displacement of a short flap, less than about 12 nt, by Pol ␦, FEN1 cleaves leaving a nick, the substrate for DNA ligase I (7-9). Because the RNA initiating the Okazaki fragments is ϳ10 nt in length, short flaps composed entirely of RNA are first displaced by Pol ␦. This does not obstruct FEN1, which is active on RNA (10, 11). In addition, displacement and cleavage occurs mostly within the first 25 nt of the downstream fragment, sufficient to remove the entire RNA/DNA primer, which is ϳ20 -30 nt in length (11). Previous biochemical studies demonstrated that primarily short flaps are cleaved by FEN1. It is likely that in vivo a series of short successive displacements by Pol ␦ and cleavages by FEN1 are effective for removal of the entire init...

Humans have five members of the well conserved RecQ helicase family: RecQ1, Bloom syndrome protein (BLM), Werner syndrome protein (WRN), RecQ4, and RecQ5, which are all known for their roles in maintaining genome stability. BLM, WRN, and RecQ4 are associated with premature aging and cancer predisposition. Of the three, RecQ4's biological and cellular roles have been least thoroughly characterized. Here we tested the helicase activity of purified human RecQ4 on various substrates. Consistent with recent results, we detected ATP-dependent RecQ4 unwinding of forked duplexes. However, our results provide the first evidence that human RecQ4's unwinding is independent of strand annealing, and that it does not require the presence of excess ssDNA. Moreover, we demonstrate that a point mutation of the conserved lysine in the Walker A motif abolished helicase activity, implying that not the N-terminal portion, but the helicase domain is solely responsible for the enzyme's unwinding activity. In addition, we demonstrate a novel stimulation of RecQ4's helicase activity by replication protein A, similar to that of RecQ1, BLM, WRN, and RecQ5. Together, these data indicate that specific biochemical activities and protein partners of RecQ4 are conserved with those of the other RecQ helicases.

The toroidal damage checkpoint complex Rad9 -Rad1-Hus1 (9-1-1) has been characterized as a sensor of DNA damage. Flap endonuclease 1 (FEN1) is a structure-specific nuclease involved both in removing initiator RNA from Okazaki fragments and in DNA repair pathways. FEN1 activity is stimulated by proliferating cell nuclear antigen (PCNA), a toroidal sliding clamp that acts as a platform for DNA replication and repair complexes. We show that 9-1-1 also binds and stimulates FEN1. Stimulation is observed on a variety of flap, nick, and gapped substrates simulating repair intermediates. Blocking 9-1-1 entry to the double strands prevents a portion of the stimulation. Like PCNA stimulation, 9-1-1 stimulation cannot circumvent the tracking mechanism by which FEN1 enters the substrate; however, 9-1-1 does not substitute for PCNA in the stimulation of DNA polymerase ␦. This suggests that 9-1-1 is a damage-specific activator of FEN1.DNA damage response ͉ DNA replication F lap endonuclease 1 (FEN1) is the primary nuclease involved in the removal of the RNA primers from Okazaki fragments (1). Deletion of the FEN1 gene in Saccharomyces cerevisiae produces temperature-sensitive growth and a phenotype common to DNA replication mutations (2-4). FEN1 is also a key nuclease in long-patch base-excision repair, a major pathway in S. cerevisiae (5-7). FEN1 cleaves a 5Ј flap substrate produced by strand-displacement synthesis during replication or repair. FEN1 cleavage activity is stimulated by proliferating cell nuclear antigen (PCNA) (8-10). In eukaryotes, long-patch base-excision repair was found to be PCNA-dependent (11-13). PCNA encircles the double-stranded region of the flap substrate and improves FEN1 binding to the cleavage site at the base of the flap (10).FEN1 has also been implicated in replication fork restart. A stalled fork can regress into a four-way junction called a chicken foot, which is structurally equivalent to a Holliday recombination junction (14,15). The regression provides a mechanism of damage repair. Werner's protein unwinds this intermediate and creates a substrate for FEN1 (16). The Werner's protein-FEN1 interaction stimulates FEN1 to cleave the strands necessary to restore replication fork structure.DNA damage evokes a cellular response that inhibits DNA replication but allows DNA repair (17). The damage response in eukaryotic cells involves activation of the ATM and ATR proteins. The ATM and ATR kinases activate checkpoint control proteins. ATM is activated in response to double-strand breaks, whereas ATR is activated in response to stalled replication forks and to a variety of damage that causes distortions and single strands (18). Rad9-Rad1-Hus1 (9-1-1) is a toroidal molecule that is loaded onto DNA by Rad17-RFC, a variation of the traditional clamp loader RFC (19). The 9-1-1 complex and ATR are recruited independently to damaged sites (20). The current model suggests that the 9-1-1 complex and ATR act as damage sensors and, therefore, participate in the activation of proteins that promote cell surviva...