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Roles of the Werner Syndrome Recq Helicase in DNA Replication

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Roles of the Werner Syndrome Recq Helicase in DNA Replication dna repair 7 (2008) 1776–1786 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/dnarepair Mini-review Roles of the Werner syndrome RecQ helicase in DNA replication Julia M. Sidorova ∗ Department of Pathology, University of Washington, Seattle, WA 98195-7705, USA article info abstract Article history: Congenital deficiency in the WRN protein, a member of the human RecQ helicase family, Received 22 July 2008 gives rise to Werner syndrome, a genetic instability and cancer predisposition disorder with Accepted 23 July 2008 features of premature aging. Cellular roles of WRN are not fully elucidated. WRN has been Published on line 6 September 2008 implicated in telomere maintenance, homologous recombination, DNA repair, and other processes. Here I review the available data that directly address the role of WRN in preserv- Keywords: ing DNA integrity during replication and propose that WRN can function in coordinating Werner syndrome replication fork progression with replication stress-induced fork remodeling. I further dis- RecQ helicase cuss this role of WRN within the contexts of damage tolerance group of regulatory pathways, Human cell culture and redundancy and cooperation with other RecQ helicases. Replication stress Published by Elsevier B.V. Damage tolerance Contents 1. Introduction ................................................................................................................. 1777 1.1. Is S phase prolonged in WRN-deficient cells? ...................................................................... 1777 1.2. What is the mechanism of the S phase extension in the absence of WRN? ...................................... 1778 1.3. Are all forks equal when it comes to WRN? ........................................................................ 1779 1.4. A mechanistic model for WRN role in replication elongation ..................................................... 1779 1.5. WRN and damage tolerance pathways ............................................................................. 1781 1.6. WRN, BLM and the question of RecQ helicase redundancy........................................................ 1782 1.7. Wrapping it all up.................................................................................................... 1782 Conflict of interest .......................................................................................................... 1783 Acknowledgments .......................................................................................................... 1783 References................................................................................................................... 1783 ∗ Tel.: +1 206 543 6585; fax: +1 206 543 3967. E-mail address: [email protected]. 1568-7864/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.dnarep.2008.07.017 dna repair 7 (2008) 1776–1786 1777 More recently, Poot et al. used immortalized lymphoblasts 1. Introduction grown in low oxygen to analyze WS cell cycle [14]. The authors used a technique of continuous BrdU labeling followed by The WRN gene is mutated in Werner syndrome (WS), a Hoechst/EthBr staining and FACS, which allows distinguish- rare autosomal recessive disorder associated with premature ing cells that divided one, two, or three times after the start aging (progeria) and predisposition to cancer [1,2]. Progeroid of labeling [15]. Applying cell cycle modeling to the data features of Werner syndrome include early onset of type (with specific assumptions such as a normal distribution of II diabetes mellitus, atherosclerosis, cataracts, skin atrophy, the probability of entering division), has yielded a conclu- graying and loss of hair, and osteoporosis. WRN is one of sion that the only two statistically significant differences the five members of the human RecQ helicase gene family, between a set of WRN−/− cells and an unrelated set of con- two other genes of which, BLM and RECQL4, are mutated in trols are a 39% extension of S phase and an increased fraction Bloom syndrome (BS) and a subset of patients with Roth- of cells permanently arrested in S phase. Later, Rodriguez- mund Thompson syndrome (RTS), respectively [3,4]. These Lopes et al. counted mitotic indices of a non-isogenic pair syndromes share the cancer predisposition feature with WS, of normal and WS primary fibroblasts grown at ambient but do not exhibit such a pronounced progeroid component. oxygen, and found that in WS cells S phase was extended Interestingly, the cell types and lineages susceptible to car- by up to 30% and the overall cell cycle by about 40%; in cinogenesis in these three syndromes are somewhat different. addition to that the fraction of dividing cells was lower WS carries an enhanced risk of neoplasms of mesenchymal [16]. origin, BS elevates the risk of the whole sporadic neoplasm We have looked at the cell cycle kinetics in pedigree spectrum, and RTS exhibits increased prevalence of osteosar- matched immortalized lymphoblast or unrelated transformed comas (reviewed in [5], also [6]). The molecular underpinnings fibroblast WS cells grown in ambient oxygen (JS unpublished). of these differences are not yet established and await a sys- WRN deficiency consistently correlated with a lower fraction tematic comparison of cellular phenotypes of the human RecQ of dividing cells. We assessed the duration of S and G2/M helicases. phases by following division of cells synchronized in late G1 Recent work has revealed that the role of WRN in human by mimosine (with and without BrdU labeling). FACS pro- pathogenesis may be broader than envisaged before, and files revealed both cell type-specific and between line-specific goes beyond heritable disease. The WRN gene is inacti- variability. Two out of three pairs of pedigree matched lym- vated by methylation in a large fraction of common sporadic phoblasts exhibited slight (less than 10%) delay in S and/or epithelial malignancies such as colon cancer, in otherwise G2/M phases in the absence of WRN, and only one out of two WRN-positive individuals [7]. Understanding molecular func- WS fibroblast lines was slower, albeit dramatically, in S phase tions of WRN thus becomes a critical task relevant both than an unrelated WRN+/+ control. to the study of human aging and the study of sporadic We thus used acute retroviral depletion of WRN to gen- carcinogenesis. erate isogenic pairs of WRN+ and WRN− cells and found Many areas of research into the functions of the human that WRN depletion from primary fibroblasts retarded S WRN RecQ helicase have been reviewed extensively (for the and/or G2/M progression and markedly reduced the fraction latest update, see [8–10]). Here, I will focus on the role of of dividing cells under standard growth conditions (ambi- human WRN in DNA replication and specifically in replication ent oxygen) [17,18]. Extension of S and/or G2/M phases in fork metabolism during stress, as caused by damage to DNA. I WRN-depleted primary fibroblasts was suppressed by lower- will revisit the data that address this problem, put these in the ing oxygen tension (Dhillon et al., submitted for publication). context of current concepts of replication stress response, and This is consistent with the observation that reducing oxygen evaluate whether a “fork-centric” view of WRN function helps tension partially suppresses the growth rate defect seen in WS arrive at a better understanding of WRN role in human cells. primary fibroblasts [19]. In contrast, WRN depletion from SV40 transformed fibrob- 1.1. Is S phase prolonged in WRN-deficient cells? lasts led to a WRN-dependent delay of cell division only when these cells were subjected to replication stress, for instance, Slow population growth in culture [11] was one of the first phe- DNA damage during S phase. The fraction of proliferating cells notypes observed in patient-derived, WRN-deficient human was not significantly affected by WRN depletion from SV40 fibroblasts maintained in ambient oxygen (20%), and it was transformed fibroblasts [18]. evident that at least two factors could be contributing to it. In summary, the data suggest that the fraction of divid- The labeling index – a percentage of cells that incorporated a ing cells can be lower in WRN-deficient cell cultures, and that pulse of 3H thymidine per specified unit of time – was lower in cells that commit to a round of division, can take more time WS cells, indicating that they entered S phase less frequently completing it. However, both of these phenotypes appear to or a larger fraction of them ceased to proliferate. In addition, be modulated by cell type, transformation status, and growth when an S phase subpopulation (identified as incorporating conditions, in particular oxygen tension. It is an open question 3H thymidine) was followed over time by monitoring appear- whether the lower dividing fraction and the extended S and/or ance of 3H-labeled mitoses, it became evident that those WS G2/M of WRN-deficient cells are linked or independent pheno- cells that entered the division cycle, spent about 10–20% more types, and in fact Szekely et al. have proposed a separate role time between two consecutive mitoses. Moreover, analysis of for WRN in counteracting oxidative damage in G1 [20]. Below these data according to Ref. [12], led to a conclusion that S I will focus on the data addressing S phase extension in WS phase was extended by about 30% in these WS fibroblasts [13]. cells. 1778 dna repair 7 (2008) 1776–1786 1.2. What is the mechanism of the S phase extension tracks with a tandem array
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