Human HEL308 localizes to damaged replication forks and unwinds lagging strand structures.

HEL308 is a superfamily II DNA helicase, conserved from archaea through to humans. HEL308 family members were originally isolated by their similarity to the Drosophila melanogaster Mus308 protein, which contributes to the repair of replication-blocking lesions such as DNA interstrand cross-links. Biochemical studies have established that human HEL308 is an ATP-dependent enzyme that unwinds DNA with a 3' to 5' polarity, but little else is know about its mechanism. Here, we show that GFP-tagged HEL308 localizes to replication forks following camptothecin treatment. Moreover, HEL308 colocalizes with two factors involved in the repair of damaged forks by homologous recombination, Rad51 and FANCD2. Purified HEL308 requires a 3' single-stranded DNA region to load and unwind duplex DNA structures. When incubated with substrates that model stalled replication forks, HEL308 preferentially unwinds the parental strands of a structure that models a fork with a nascent lagging strand, and the unwinding action of HEL308 is specifically stimulated by human replication protein A. Finally, we show that HEL308 appears to target and unwind from the junction between single-stranded to double-stranded DNA on model fork structures. Together, our results suggest that one role for HEL308 at sites of blocked replication might be to open up the parental strands to facilitate the loading of subsequent factors required for replication restart.

Mph1 requires mismatch repair-independent and -dependent functions of MutSalpha to regulate crossover formation during homologous recombination repair.

In budding yeast the DNA helicase Mph1 prevents genome rearrangements during ectopic homologous recombination (HR) by suppressing the formation of crossovers (COs). Here we show that during ectopic HR repair, the anti-CO function of Mph1 is intricately associated with the mismatch repair (MMR) factor, MutSalpha. In particular, during HR repair using a completely homologous substrate, we reveal an MMR-independent function of MutSalpha in generating COs that is specifically antagonized by Mph1, but not Sgs1. In contrast, both Mph1 and MutSalpha are required to efficiently suppress COs in the presence of a homeologous substrate. Mph1 acts redundantly with Sgs1 in this respect since mph1Delta sgs1Delta double mutant cells pheno-copy MutSalpha mutants and completely fail to discriminate homologous and homeologous sequences during HR repair. However, this defect of mph1Delta sgs1Delta cells is not due to an inability to carry out MMR but rather is accompanied by elevated levels of gene conversion (GC) and bi-directional GC tracts specifically in non-crossover products. Models describing how Mph1, MutSalpha and Sgs1 act in concert to suppress genome rearrangements during ectopic HR repair are discussed.

Overlapping roles for Yen1 and Mus81 in cellular Holliday junction processing.

Eukaryotic Holliday junction (HJ) resolvases have attracted much attention recently with the identification of at least three distinct proteins that can cleave model HJs in vitro. However, the specific DNA structure(s) that these proteins act upon in the cell is unknown. Here, we describe a system in budding yeast to directly and quantitatively monitor in vivo HJ resolution. We found that Yen1 acts redundantly with Mus81, but not Slx1, to resolve a model HJ in vivo. This functional overlap specifically extends to the repair/bypass of lesions that impede the progression of replication forks but not to the repair of double-strand breaks induced by ionizing radiation. Together, these results suggest a direct role for Yen1 in the response to DNA damage and implicate overlapping HJ resolution functions of Yen1 with Mus81 during replication fork repair.

Remodeling of DNA replication structures by the branch point translocase FANCM.

Fanconi anemia (FA) is a genetically heterogeneous chromosome instability syndrome associated with congenital abnormalities, bone marrow failure, and cancer predisposition. Eight FA proteins form a nuclear core complex, which promotes tolerance of DNA lesions in S phase, but the underlying mechanisms are still elusive. We reported recently that the FA core complex protein FANCM can translocate Holliday junctions. Here we show that FANCM promotes reversal of model replication forks via concerted displacement and annealing of the nascent and parental DNA strands. Fork reversal by FANCM also occurs when the lagging strand template is partially single-stranded and bound by RPA. The combined fork reversal and branch migration activities of FANCM lead to extensive regression of model replication forks. These observations provide evidence that FANCM can remodel replication fork structures and suggest a mechanism by which FANCM could promote DNA damage tolerance in S phase.

The Bloom's syndrome helicase suppresses crossing over during homologous recombination.

Mutations in BLM, which encodes a RecQ helicase, give rise to Bloom's syndrome, a disorder associated with cancer predisposition and genomic instability. A defining feature of Bloom's syndrome is an elevated frequency of sister chromatid exchanges. These arise from crossing over of chromatid arms during homologous recombination, a ubiquitous process that exists to repair DNA double-stranded breaks and damaged replication forks. Whereas crossing over is required in meiosis, in mitotic cells it can be associated with detrimental loss of heterozygosity. BLM forms an evolutionarily conserved complex with human topoisomerase IIIalpha (hTOPO IIIalpha), which can break and rejoin DNA to alter its topology. Inactivation of homologues of either protein leads to hyper-recombination in unicellular organisms. Here, we show that BLM and hTOPO IIIalpha together effect the resolution of a recombination intermediate containing a double Holliday junction. The mechanism, which we term double-junction dissolution, is distinct from classical Holliday junction resolution and prevents exchange of flanking sequences. Loss of such an activity explains many of the cellular phenotypes of Bloom's syndrome. These results have wider implications for our understanding of the process of homologous recombination and the mechanisms that exist to prevent tumorigenesis.

Wrestling off RAD51: a novel role for RecQ helicases.

Homologous recombination (HR) is essential for the accurate repair of DNA double-strand breaks and damaged replication forks. However, inappropriate or aberrant HR can also result in genome rearrangements. The maintenance of cell viability is, therefore, a careful balancing act between the benefits of HR (the error-free repair of DNA strand breaks) and the potential detrimental outcomes of HR (chromosomal rearrangements). Two papers have recently provided a mechanistic insight into how HR may be tempered by RecQ helicases to prevent genome instability and diseases that are a consequence of this, such as cancer.

Role of the BLM helicase in replication fork management.

Genomic DNA is particularly vulnerable to mutation during S-phase when the two strands of parental duplex DNA are separated during the process of semi-conservative DNA replication. Lesions that are normally repaired efficiently in the context of double stranded DNA can cause replication forks to stall or, more dangerously, collapse. Cells from Bloom's syndrome patients, that lack the RecQ helicase BLM, show defects in the response to replicative stress and contain a multitude of chromosomal aberrations, which primarily arise through excessive levels of homologous recombination. Here, recent findings are reviewed that further our understanding of the role that BLM plays in the management of damaged replication forks.

The Bloom's syndrome helicase promotes the annealing of complementary single-stranded DNA.

The product of the gene mutated in Bloom's syndrome, BLM, is a 3'-5' DNA helicase belonging to the highly conserved RecQ family. In addition to a conventional DNA strand separation activity, BLM catalyzes both the disruption of non-B-form DNA, such as G-quadruplexes, and the branch migration of Holliday junctions. Here, we have characterized a new activity for BLM: the promotion of single-stranded DNA (ssDNA) annealing. This activity does not require Mg(2+), is inhibited by ssDNA binding proteins and ATP, and is dependent on DNA length. Through analysis of various truncation mutants of BLM, we show that the C-terminal domain is essential for strand annealing and identify a 60 amino acid stretch of this domain as being important for both ssDNA binding and strand annealing. We present a model in which the ssDNA annealing activity of BLM facilitates its role in the processing of DNA intermediates that arise during repair of damaged replication forks.

The Bloom's syndrome helicase stimulates the activity of human topoisomerase IIIalpha.

Bloom's syndrome (BS) is a disorder associated with chromosomal instability and a predisposition to the development of cancer. The BS gene product, BLM, is a DNA helicase of the RecQ family that forms a complex in vitro and in vivo with topoisomerase IIIalpha. Here, we show that BLM stimulates the ability of topoisomerase IIIalpha to relax negatively supercoiled DNA. Moreover, DNA binding analyses indicate that BLM recruits topoisomerase IIIalpha to its DNA substrate. Consistent with this, a mutant form of BLM that retains helicase activity, but is unable to bind topoisomerase IIIalpha, fails to stimulate topoisomerase activity. These results indicate that a physical association between BLM and topoisomerase IIIalpha is a prerequisite for their functional biochemical interaction.

RecQ helicases and cellular responses to DNA damage.

The faithful replication of the genome is essential for the survival of all organisms. It is not surprising therefore that numerous mechanisms have evolved to ensure that duplication of the genome occurs with only minimal risk of mutation induction. One mechanism of genome destabilization is replication fork demise, which can occur when a translocating fork meets a lesion or adduct in the template. Indeed, the collapse of replication forks has been suggested to occur in every replicative cell cycle making this a potentially significant problem for all proliferating cells. The RecQ helicases, which are essential for the maintenance of genome stability, are thought to function during DNA replication. In particular, RecQ helicase mutants display replication defects and have phenotypes consistent with an inability to efficiently reinitiate replication following replication fork demise. Here, we review some current models for how replication fork repair might be effected, and discuss potential roles for RecQ helicases in this process.

A challenging diagnosis: case report of extensive pyoderma gangrenosum at multiple sites.

BACKGROUND: Pyoderma gangrenosum (PG) is a rare dermatological condition characterized by the rapid progression of a painful, necrolytic ulcer with an irregular, undermined border and commonly affects the lower extremities, mainly in the pretibial area. The diagnosis of PG is not easy. Due to lack of diagnostic laboratory test and histopathological findings indicative of PG, it is often misdiagnosed as an infection. This results in delayed or inappropriate treatment of the condition, which leads to devastating consequences such as limb amputation and death. MAIN OBSERVATIONS: We report a rare case of a 51-year-old female who was initially diagnosed as having infected ulcers and underwent serial debridements, which resulted in extensive PG at three different sites (abdominal, left thigh, and sacral). CONCLUSION: This case highlights the challenges in diagnosing PG, emphasizes the key clinical features to aid diagnosis, and the clinical consequences of delayed or misdiagnosis of this condition.

DNA helicases required for homologous recombination and repair of damaged replication forks.

DNA helicases are found in all kingdoms of life and function in all DNA metabolic processes where the two strands of duplex DNA require to be separated. Here, we review recent developments in our understanding of the roles that helicases play in the intimately linked processes of replication fork repair and homologous recombination, and highlight how the cell has evolved many distinct, and sometimes antagonistic, uses for these enzymes.

The Bloom's syndrome helicase can promote the regression of a model replication fork.

Homozygous inactivation of BLM gives rise to Bloom's syndrome, a disorder associated with genomic instability and cancer predisposition. BLM encodes a member of the RecQ DNA helicase family that is required for the maintenance of genome stability and the suppression of sister-chromatid exchanges. BLM has been proposed to function in the rescue of replication forks that have collapsed or stalled as a result of encountering lesions that block fork progression. One proposed mechanism of fork rescue involves regression in which the nascent leading and lagging strands anneal to create a so-called "chicken foot" structure. Here we have developed an in vitro system for analysis of fork regression and show that BLM, but not Escherichia coli RecQ, can promote the regression of a model replication fork. BLM-mediated fork regression is ATP-dependent and occurs processively, generating regressed arms of >250 bp in length. These data establish the existence of a eukaryotic protein that could promote replication fork regression in vivo and suggest a novel pathway through which BLM might suppress genetic exchanges.

BLAP75/RMI1 promotes the BLM-dependent dissolution of homologous recombination intermediates.

BLM encodes a member of the highly conserved RecQ DNA helicase family, which is essential for the maintenance of genome stability. Homozygous inactivation of BLM gives rise to the cancer predisposition disorder Bloom's syndrome. A common feature of many RecQ helicase mutants is a hyperrecombination phenotype. In Bloom's syndrome, this phenotype manifests as an elevated frequency of sister chromatid exchanges and interhomologue recombination. We have shown previously that BLM, together with its evolutionarily conserved binding partner topoisomerase IIIalpha (hTOPO IIIalpha), can process recombination intermediates that contain double Holliday junctions into noncrossover products by a mechanism termed dissolution. Here we show that a recently identified third component of the human BLM/hTOPO IIIalpha complex, BLAP75/RMI1, promotes dissolution catalyzed by hTOPO IIIalpha. This activity of BLAP75/RMI1 is specific for dissolution catalyzed by hTOPO IIIalpha because it has no effect in reactions containing either Escherichia coli Top1 or Top3, both of which can also catalyze dissolution in a BLM-dependent manner. We present evidence that BLAP75/RMI1 acts by recruiting hTOPO IIIalpha to double Holliday junctions. Implications of the conserved ability of type IA topoisomerases to catalyze dissolution and how the evolution of factors such as BLAP75/RMI1 might confer specificity on the execution of this process are discussed.

The HRDC domain of BLM is required for the dissolution of double Holliday junctions.

Bloom's syndrome is a hereditary cancer-predisposition disorder resulting from mutations in the BLM gene. In humans, BLM encodes one of five members of the RecQ helicase family. One function of BLM is to act in concert with topoisomerase IIIalpha (TOPO IIIalpha) to resolve recombination intermediates containing double Holliday junctions by a process called double Holliday junction dissolution, herein termed dissolution. Here, we show that dissolution is highly specific for BLM among human RecQ helicases and critically depends upon a functional HRDC domain in BLM. We show that the HRDC domain confers DNA structure specificity, and is required for the efficient binding to and unwinding of double Holliday junctions, but not for the unwinding of a simple partial duplex substrate. Furthermore, we show that lysine-1270 of BLM, which resides in the HRDC domain and is predicted to play a role in mediating interactions with DNA, is required for efficient dissolution.

Vascular endothelial growth factor expression and glomerular endothelial cell loss in the remnant kidney model.

BACKGROUND: Vascular endothelial growth factor (VEGF) is constitutively expressed in the glomerulus where it may have a role in the maintenance of capillary endothelial cell integrity. The present study sought to examine changes in VEGF expression in a model of progressive renal disease and to assess the effects of angiotensin converting enzyme (ACE) inhibition. METHODS: Subtotal nephrectomized (STNx) rats were randomly assigned to receive vehicle (n=10) or the ACE inhibitor perindopril (8 mg/l drinking water) for 12 weeks duration (n=10). Sham-operated rats were used as controls (n=10). Glomerular capillary endothelial cell density was evaluated by immunostaining for the pan-endothelial cell marker RECA-1 and VEGF expression was assessed by quantitative in situ hybridization. RESULTS: In STNx rats glomerular capillary endothelial cell density was reduced to 19% that of sham rats (P<0.01) with a concomitant reduction in glomerular VEGF expression, also to 19% of sham rats (P<0.01). Perindopril treatment was associated with normalization of both capillary endothelial cell density and glomerular VEGF mRNA. CONCLUSIONS: Reduction in glomerular VEGF expression is a feature of the renal pathology that follows subtotal nephrectomy. In the context of the known functions of this growth factor, these findings suggest that diminution in VEGF may contribute to the demonstrated loss of glomerular endothelium that develops in this model of progressive renal disease.

Stimulation of flap endonuclease-1 by the Bloom's syndrome protein.

Bloom's syndrome (BS) is a rare autosomal recessive genetic disorder associated with genomic instability and an elevated risk of cancer. Cellular features of BS include an accumulation of abnormal replication intermediates and increased sister chromatid exchange. Although it has been suggested that the underlying defect responsible for hyper-recombination in BS cells is a temporal delay in the maturation of DNA replication intermediates, the precise role of the BS gene product, BLM, in DNA metabolism remains elusive. We report here a novel interaction of the BLM protein with the human 5'-flap endonuclease/5'-3' exonuclease (FEN-1), a genome stability factor involved in Okazaki fragment processing and DNA repair. BLM protein stimulates both the endonucleolytic and exonucleolytic cleavage activity of FEN-1 and this functional interaction is independent of BLM catalytic activity. BLM and FEN-1 are associated with each other in human nuclei as shown by their reciprocal co-immunoprecipitation from HeLa nuclear extracts. The BLM-FEN-1 physical interaction is mediated through a region of the BLM C-terminal domain that shares homology with the FEN-1 interaction domain of the Werner syndrome protein, a RecQ helicase family member homologous to BLM. This study provides the first evidence for a direct interaction of BLM with a human nucleolytic enzyme. We suggest that functional interactions between RecQ helicases and Rad2 family nucleases serve to process DNA substrates that are intermediates in DNA replication and repair.

Functional interaction between the Bloom's syndrome helicase and the RAD51 paralog, RAD51L3 (RAD51D).

Bloom's syndrome (BS) is a genetic disorder associated with short stature, fertility defects, and a predisposition to the development of cancer. BS cells are characterized by genomic instability; in particular, a high rate of reciprocal exchanges between sister-chromatids and homologous chromosomes. The BS gene product, BLM, is a helicase belonging to the highly conserved RecQ family. BLM is known to form a complex with the RAD51 recombinase, and to act upon DNA intermediates that form during homologous recombination, including D-loops and Holliday junctions. Here, we show that BLM also makes a direct physical association with the RAD51L3 protein (also known as RAD51D), a so-called RAD51 paralog that shows limited sequence similarity to RAD51 itself. This interaction is mediated through the N-terminal domain of BLM. To analyze functional interactions between BLM and RAD51L3, we have purified a heteromeric complex comprising RAD51L3 and a second RAD51 paralog, XRCC2. We show that the RAD51L3-XRCC2 complex stimulates BLM to disrupt synthetic 4-way junctions that model the Holliday junction. We also show that a truncated form of BLM, which retains helicase activity but is unable to bind RAD51L3, is not stimulated by the RAD51L3-XRCC2 complex. Our data indicate that the activity of BLM is modulated through an interaction with the RAD51L3-XRCC2 complex, and that this stimulatory effect on BLM is dependent upon a direct physical association between the BLM and RAD51L3 proteins. We propose that BLM co-operates with RAD51 paralogs during the late stages of homologous recombination processes that serve to restore productive DNA replication at sites of damaged or stalled replication forks.