Elsevier

DNA Repair

Volume 10, Issue 4, 3 April 2011, Pages 416-426
DNA Repair

Depletion of the bloom syndrome helicase stimulates homology-dependent repair at double-strand breaks in human chromosomes

https://doi.org/10.1016/j.dnarep.2011.01.009Get rights and content

Abstract

Mutation of BLM helicase causes Blooms syndrome, a disorder associated with genome instability, high levels of sister chromatid exchanges, and cancer predisposition. To study the influence of BLM on double-strand break (DSB) repair in human chromosomes, we stably transfected a normal human cell line with a DNA substrate that contained a thymidine kinase (tk)-neo fusion gene disrupted by the recognition site for endonuclease I-SceI. The substrate also contained a closely linked functional tk gene to serve as a recombination partner for the tk-neo fusion gene. We derived two cell lines each containing a single integrated copy of the DNA substrate. In these cell lines, a DSB was introduced within the tk-neo fusion gene by expression of I-SceI. DSB repair events that occurred via homologous recombination (HR) or nonhomologous end-joining (NHEJ) were recovered by selection for G418-resistant clones. DSB repair was examined under conditions of either normal BLM expression or reduced BLM expression brought about by RNA interference. We report that BLM knockdown in both cell lines specifically increased the frequency of HR events that produced deletions by crossovers or single-strand annealing while leaving the frequency of gene conversions unchanged or reduced. We observed no change in the accuracy of individual HR events and no substantial alteration of the nature of individual NHEJ events when BLM expression was reduced. Our work provides the first direct evidence that BLM influences DSB repair pathway choice in human chromosomes and suggests that BLM deficiency can engender genomic instability by provoking an increased frequency of HR events of a potentially deleterious nature.

Introduction

The genome of a mammalian cell must contend with a variety of types of DNA damage every day [1], [2]. One potentially serious threat is the DNA double-strand break (DSB). DSBs can arise either directly or indirectly from exposure to chemical or radiological agents, from metabolism of spontaneous lesions, or at stalled replication forks. It is imperative that DSBs be dealt with efficiently and accurately to avoid detrimental chromosomal rearrangements. There are two broad classes of DSB repair pathways that operate in mammalian cells: homologous recombination (HR), and nonhomologous end-joining (NHEJ) (recently reviewed in [3–6]). HR classically involves the use of a DNA template to maintain or restore genetic information, and so HR is considered to be an accurate means of DSB repair. In contrast, NHEJ is untemplated and is considered to be error-prone since it is often accompanied by deletion of sequence at the site of the healed DSB. Both HR and NHEJ are important pathways and defects in either of these pathways can lead to genomic instability and disease [3], [4]. HR is active primarily during late S and G2 phases of the cell cycle, while NHEJ appears to be active throughout the cell cycle (reviewed in [7]). There remain important gaps in our understanding of how the choice is made between the HR and NHEJ pathways for DSB repair, although recent work suggests that regulation of 5′–3′ resection of DNA ends, an early step in HR, may play an important role in pathway choice [8], [9], [10], [11].

Although HR is generally considered to be accurate, there are potential hazards involved in the execution of HR that must be avoided in order for genome integrity to be preserved. The use of inappropriate recombination partners, for example, can lead to chromosomal rearrangements such as translocations and deletions. HR events, particularly those resolving as crossovers, can also be detrimental merely by occurring at too high a rate since high levels of such events will likely increase the incidence of loss of heterozygosity (LOH). LOH has been implicated in carcinogenesis by facilitating the loss of functional copies of tumor suppressor genes [12], [13], [14]. In addition, a distinct “nonconservative” form of HR, known as single-strand annealing (SSA), does not involve the use of a DNA template and can generate deletions via the splicing together of repeated homologous sequences with the concomitant loss of sequence between the repeats [3], [6]. To maintain genome stability, DSB repair in its varied forms must therefore be appropriately regulated qualitatively and quantitatively.

One protein that has gained some attention in recent years as a regulator of HR is the Bloom syndrome helicase (BLM). BLM helicase is a member of the RecQ family of DNA helicases, a highly conserved family of enzymes named for the prototypical RecQ helicase from E. coli. Five DNA helicases of the RecQ family (BLM, WRN, RecQL, RecQ4, and RecQ5), have been identified in humans. Mutations in BLM, WRN and RecQ4 lead to Bloom, Werner, and Rothmund-Thomson syndromes, respectively. Patients with Bloom syndrome (BS) display growth defects, genomic instability (including translocations and quadriradials), and a strong predisposition to a broad spectrum of cancers [15]. One hallmark of BS cells is a greatly elevated frequency of sister chromatid exchanges (SCE) [15], [16]. Numerous studies of the role played by BLM helicase in maintaining genome stability have collectively revealed multiple roles for BLM in HR regulation [17], [18], [19], [20], [21], [22], [23]. Curiously, both pro-HR and anti-HR functions have been assigned to BLM.

In terms of pro-HR activities, BLM reportedly promotes 5′–3′ strand resection at DSB ends [9], [10], a processing step necessary for the initiation of HR. It has also been reported recently [24] that BLM may act early in HR by stimulating the DNA strand exchange activity of Rad51. Annealing of complementary strands of DNA is another activity in BLM's repertoire [25]. BLM has been implicated in promoting regression of stalled replication forks to produce a structural configuration conducive to HR-dependent restart of replication [26], [27]. BLM has additionally been shown to catalyze branch migration of Holliday junction recombination intermediates [28]. In conjunction with BLAP75 and topoisomerase IIIα, BLM has been shown to potentially play a role in late steps of HR by catalyzing what has been termed double Holliday junction (DHJ) dissolution [29], [30], [31]. In DHJ dissolution, which to date has been demonstrated only in vitro using substrates that model a DHJ recombination intermediate, two Holliday junctions are first converged by BLM and then decatenated by topoisomerase IIIα to separate the two DNA duplexes that are linked by the DHJ recombination intermediate. Dissolution leads exclusively to noncrossover recombination products. An alternative to DHJ dissolution is the process of resolution, an endonucleolytic cleavage of the DHJ that can produce both crossovers and noncrossovers [5]. BLM's role in DHJ dissolution may normally serve to suppress LOH and SCE, and may help explain the elevated levels of SCE seen in cells from BS patients [15], [16] and the high levels of LOH and SCE seen in mouse and human cells experimentally depleted of BLM [32], [33], [34].

In terms of potential anti-HR activities, BLM can disrupt the D-loop structure formed following the initial strand invasion step of HR [35], [36]. Although this activity may serve as a mechanism to avoid the use of inappropriate templates, it is possible that under certain circumstances displacement of the invading strand from the D-loop by BLM may actually promote HR via the synthesis-dependent stand annealing (SDSA) pathway [37], [38], [39] which proceeds without the formation of Holliday junction intermediates. In SDSA, one or both 3′ ends from a DSB invades an homologous template, and DNA synthesis is primed from the invading end. The newly synthesized strand is then displaced from the template and anneals with complementary sequence from the other end of the DSB. Notably, the SDSA pathway results almost exclusively in noncrossovers [40]. BLM has also been shown to disrupt Rad51 nucleofilaments, but only when the filament is in its inactive ADP-bound form [38]. Thus, BLM may wield a type of “quality control” over HR by disrupting inappropriate events at a very early stage. Finally, the ability of BLM to anneal complementary strands as well as catalyze branch migration of Holliday junctions may implicate BLM not only in the regression of stalled replication forks but also in subsequent reversal of regressed forks. By undoing the so-called “chicken-foot” structure produced by fork regression, BLM may actually prevent recombination events at stalled replication forks [28].

Although it is clear that BLM helicase is an important regulator of HR, more work is needed to sort out BLM's varied and seemingly disparate roles. Perhaps surprisingly, most previous work on human BLM has been carried out using cell-free or plasmid-based systems while comparatively few studies have been carried out on the impact of BLM on DNA transactions as they occur within the chromosomes of living human cells. We have developed an experimental system that allows the study of DSB repair, at the nucleotide level, within the genome of mitotically dividing human cells. Our system involves the integration of a DNA substrate engineered so that DNA transactions may restore function to a selectable marker gene. Expression of endonuclease I-SceI is used to deliver a specific DSB to the genome within the integrated substrate and we can investigate repair of the DSB carried out by HR or NHEJ.

Using our system in conjunction with an RNA interference approach to knockdown expression of BLM, we investigated the role of BLM in DSB repair in cultured human fibroblasts. We report here that knockdown of BLM in human cells altered genomic DSB repair by bringing about a significant shift toward repair via HR that produced deletions either by crossovers or by SSA. Our work implicates BLM as an important participant in DSB pathway choice and provides the first direct evidence that BLM deficiency may contribute to genome instability in somatic human cells by stimulating a specific type of HR event.

Section snippets

Cell lines and DSB repair substrate

Cell line pLB4/11, described previously [41], [42], and cell line pLB4/20 were derived from SV40-immortalized normal human fibroblast cell line GM637 (obtained from the NIGMS) and each line contains a single integrated copy of recombination substrate pLB4 (Fig. 1). pLB4 contains a tk-neo fusion gene disrupted by the insertion of a 22 bp oligonucleotide that contains the 18-bp recognition sequence for yeast endonuclease I-SceI. In addition, pLB4 contains a 2.5 kb HindIII fragment containing a

A system for monitoring intrachromosomal DSB repair events in human cells

The centerpiece of our strategy is substrate pLB4 [41], [42] shown in Fig. 1. A single copy of pLB4 was stably integrated in the genome of normal human fibroblast cell line GM637 to independently derive cell lines pLB4/11 and pLB4/20. A seminal feature of pLB4 is a tk-neo fusion gene disrupted by a 22 bp oligonucleotide inserted into the tk portion of the fusion gene. The oligonucleotide contains the 18 bp recognition site for yeast endonuclease I-SceI. Also contained on pLB4 is a complete,

Discussion

In this paper we report the first direct evidence that the level of expression of BLM helicase influences the manner in which a DSB is repaired in the genome of human cells. More specifically, BLM depletion significantly increased the frequency of homology-dependent DSB repair events that engendered chromosomal deletion.

The loss of BLM activity in the rare genetic disorder BS leads to genomic instability and a strong predisposition to a wide spectrum of cancers. BLM defects have also been

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by Public Health Service grant GM081472 from the National Institute for General Medical Sciences to A.S.W. We thank members of the Waldman lab for helpful discussions.

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