Elsevier

DNA Repair

Volume 12, Issue 2, 1 February 2013, Pages 128-139
DNA Repair

Distinct functions of human RECQ helicases WRN and BLM in replication fork recovery and progression after hydroxyurea-induced stalling

https://doi.org/10.1016/j.dnarep.2012.11.005Get rights and content

Abstract

Human WRN and BLM genes are members of the conserved RECQ helicase family. Mutations in these genes are associated with Werner and Bloom syndromes. WRN and BLM proteins are implicated in DNA replication, recombination, repair, telomere maintenance, and transcription. Using microfluidics-assisted display of DNA for replication track analysis (ma-RTA), we show that WRN and BLM contribute additively to normal replication fork progression, and non-additively, in a RAD51-dependent pathway, to resumption of replication after arrest by hydroxyurea (HU), a replication-stalling drug. WRN but not BLM is required to support fork progression after HU. Resumption of replication by forks may be necessary but is not sufficient for timely completion of the cell cycle after HU arrest, as depletion of WRN or BLM compromises fork recovery to a similar degree, but only BLM depletion leads to extensive delay of cell division after HU, as well as more pronounced chromatin bridging. Finally, we show that recovery from HU includes apparent removal of some of the DNA that was synthesized immediately after release from HU, a novel phenomenon that we refer to as nascent strand processing, NSP.

Highlights

► WRN and BLM human RECQ helicases contribute additively to fork progression during normal replication. ► After short-term HU block, fork restart requires non-additive contributions of BLM, WRN, and RAD51 that do not involve protection from DSBs. ► BLM but not WRN is required for timely G2/M transition after short-term HU block. ► Human fibroblasts appear to replace or degrade some of the DNA synthesized by recovering forks during the first hour after release from HU.

Introduction

RECQ helicases are a family of proteins conserved from bacteria to humans. Out of five human RECQ helicase genes, three are associated with heritable disorders. Mutations in the BLM [1], and WRN [2] genes cause, respectively, Bloom syndrome (BS) and Werner syndrome (WS), and mutations in RECQL4 [3] are seen in Rothmund–Thomson, RAPADILINO, and Baller-Gerold (BGS) syndromes.

Clinical manifestations of Werner syndrome mimic premature aging, while Bloom syndrome is associated with developmental abnormalities [4]. Bloom and Werner syndromes are cancer-prone diseases, albeit the spectra of cancers they predispose to are different. Cells mutated in BLM or WRN genes show phenotypes associated with genomic instability and perturbed replication: slower S phase, increased fraction of cells at the G2/M boundary of the cell cycle, and expression of some fragile sites (for review, see [5], [6], [7], [8]). In vitro, several biochemical features are unique to BLM or WRN, warranting a systematic analysis of the redundancy and cooperation between these two RECQs within a cell. Studies in DT40 cells demonstrated synthetic hypersensitivity of WRN/BLM knock-out cells to a number of genotoxic drugs, including camptothecin [9], as well as unique genetic interactions between these RECQs and other genes [10], pointing toward WRN and BLM's complementary roles within pathways of DNA metabolism, and inviting a more mechanistic inquiry.

The insight into roles of WRN and BLM in DNA replication is complicated by the facts that both RECQs are multifunctional proteins [4], [11], and that replication fork metabolism is likely conducted through several interconnected pathways [8], [12]. Briefly, when fork progression is interrupted by lesions in the template or by replisome poisoning, extra activities are turned on as part of the S phase checkpoint, and stabilize the replisome-DNA structure against collapse [12], [13]. It is thought that collapsed replication forks are susceptible to double strand breaks (DSBs). These DSBs may be an intermediate in an active fork rescue pathway, or merely a breakdown product which necessitates repair (see Refs. above). The exact balance between fork stabilization and fork collapse/rescue may depend on the cell type and the nature of interruption facing a fork.

Early studies have suggested that both WRN and BLM can be involved in elongation of DNA replication (reviewed in [8]). The use of DNA fiber technology allowed further insight into roles of RECQ helicases at a replication fork, demonstrating that WRN [14] and BLM [15] may be required for normal fork progression. In addition, complementing BS patient-derived human fibroblasts with BLM improves resumption of replication fork progression after an arrest with hydroxyurea (HU), a ribonucleotide reductase inhibitor [16]. Defects of fork recovery, albeit variable, were also demonstrated in WRN-depleted HeLa cells, in WS fibroblasts [17], [18], and in WRN-depleted fibroblasts [19]. Both RECQs are targeted by the checkpoint kinase ATR [18], [20], [21], [22] and affect checkpoint performance [16], [23], [24].

In order to delineate redundant versus cooperative functions of WRN and BLM, we have established isogenic human fibroblasts depleted of WRN, BLM, or both RECQs [25]. Here, we undertake a detailed analysis of replication fork phenotypes in these cells, and describe both unique and shared functions of WRN and BLM at a replication fork, as well as uncover a novel process of metabolizing nascent strands during recovery from HU.

Section snippets

Cells and culture

SV40-transformed GM639 fibroblast cell line was obtained from the Coriell Institute Cell Repositories (Camden NJ). GM639cc1 is a pNeoA derivative of GM639 [19], [25], [26]. Unless stated otherwise, all experiments were performed using this cell line. The large T antigen is at least partially inactivated in this cell line since it does not support replication of SV40 origin-containing plasmids (J.S., unpub.).

The primary human dermal fibroblasts were described [27]. All cell lines were grown in

WRN and BLM contribute additively to fork progression rates during an unperturbed S phase

We depleted WRN and/or BLM with lentiviral shRNAs, as before ([19], [25] and Fig. 4A and Figs. S1A, S3C and S4B), achieving at least 80–85% depletion of the target protein(s). Growth rate was lower in WRN-depleted and, more dramatically, in BLM-depleted cell populations, which reflected the size of replicating fraction. To account for it, every assay used in this study discriminated between replicating and non-replicating fractions, or focused exclusively on replicating fraction.

We labeled

WRN and BLM contribute additively to fork progression during unstressed replication

BLM-deficient cells exhibit slowed fork progression [15], while WRN-deficient cells have an increased level of asymmetrically diverging forks in early S phase, suggesting fork inactivation [14]. Our study confirmed that BLM, and to a lesser degree, WRN, are needed for normal fork progression, and we showed for the first time that when both RECQs were depleted, fork progression was slower than in single-depleted cells. Thus, BLM and WRN can partially substitute for each other or perform parallel

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

We are grateful to Drs. Bonita Brewer, M.K. Raghuraman, and members of their lab for support and discussions, and to Drs. Wenyi Feng and Katharina Schlacher for communicating unpublished results and for critical discussions. Thanks are due to Dr. Babak Parviz and Ehsan Saeedi for manufacturing a microchannel template, to Dr. Albert Folch and his lab for the use of Plasma Preen, and to Alden Hackmann for help with figures. This work was supported by an NCI P01 grant CA77852 to R.M. Jr.

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