Mechanisms by which Bloom protein can disrupt recombination intermediates of Okazaki fragment maturation

Bloom syndrome is a familial genetic disorder associated with sunlight sensitivity and a high predisposition to cancers. The mutated gene, Bloom protein (BLM), encodes a DNA helicase that functions in genome maintenance via roles in recombination repair and resolution of recombination structures. We designed substrates representing illegitimate recombination intermediates formed when a displaced DNA flap generated during maturation of Okazaki fragments escapes cleavage by flap endonuclease-1 and anneals to a complementary ectopic DNA site. Results show that displaced, replication protein A (RPA)-coated flaps could readily bind and ligate at the complementary site to initiate recombination. RPA also displayed a strand-annealing activity that hastens the rate of recombination intermediate formation. BLM helicase activity could directly disrupt annealing at the ectopic site and promote flap endonuclease-1 cleavage. Additionally, BLM has its own strand-annealing and strand-exchange activities. RPA inhibited the BLM strand-annealing activity, thereby promoting helicase activity and complex dissolution. BLM strand exchange could readily dissociate invading flaps, e.g. in a D-loop, if the exchange step did not involve annealing of RPA-coated strands. Use of ATP to activate the helicase function did not aid flap displacement by exchange, suggesting that this is a helicase-independent mechanism of complex dissociation. When RPA could bind, it displayed its own strand-exchange activity. We interpret these results to explain how BLM is well equipped to deal with alternative recombination intermediate structures.     

On the roles of Saccharomyces cerevisiae Dna2p and Flap endonuclease 1 in Okazaki fragment processing

Short DNA segments designated Okazaki fragments are intermediates in eukaryotic DNA replication. Each contains an initiator RNA/DNA primer (iRNA/DNA), which is converted into a 5'-flap and then removed prior to fragment joining. In one model for this process, the flap endonuclease 1 (FEN1) removes the iRNA. In the other, the single-stranded binding protein, replication protein A (RPA), coats the flap, inhibits FEN1, but stimulates cleavage by the Dna2p helicase/nuclease. RPA dissociates from the resultant short flap, allowing FEN1 cleavage. To determine the most likely process, we analyzed cleavage of short and long 5'-flaps. FEN1 cleaves 10-nucleotide fixed or equilibrating flaps in an efficient reaction, insensitive to even high levels of RPA or Dna2p. On 30-nucleotide fixed or equilibrating flaps, RPA partially inhibits FEN1. CTG flaps can form foldback structures and were inhibitory to both nucleases, however, addition of a dT(12) to the 5'-end of a CTG flap allowed Dna2p cleavage. The presence of high Dna2p activity, under reaction conditions favoring helicase activity, substantially stimulated FEN1 cleavage of tailed-foldback flaps and also 30-nucleotide unstructured flaps. Our results suggest Dna2p is not used for processing of most flaps. However, Dna2p has a role in a pathway for processing structured flaps, in which it aids FEN1 using both its nuclease and helicase activities.     

Components of the Secondary Pathway Stimulate the Primary Pathway of Eukaryotic Okazaki Fragment Processing

Reconstitution of eukaryotic Okazaki fragment processing implicates both one- and two-nuclease pathways for processing flap intermediates. In most cases, FEN1 (flap endonuclease 1) is able to efficiently cleave short flaps as they form. However, flaps escaping cleavage bind replication protein A (RPA) inhibiting FEN1. The flaps must then be cleaved by Dna2 nuclease/helicase before FEN1 can act. Pif1 helicase aids creation of long flaps. The pathways were considered connected only in that the products of Dna2 cleavage are substrates for FEN1. However, results presented here show that Dna2, Pif1, and RPA, the unique proteins of the two-nuclease pathway from Saccharomyces cerevisiae, all stimulate FEN1 acting in the one-nuclease pathway. Stimulation is observed on RNA flaps representing the initial displacement and on short DNA flaps, subsequently displaced. Neither the RNA nor the short DNA flaps can bind the two-nuclease pathway proteins. Instead, direct interactions between FEN1 and the two-nuclease pathway proteins have been detected. These results suggest that the proteins are either part of a complex or interact successively with FEN1 because the level of stimulation would be similar either way. Proteins bound to FEN1 could be tethered to the flap base by the interaction of FEN1 with PCNA, potentially improving their availability when flaps become long. These findings also support a model in which cleavage by FEN1 alone is the preferred pathway, with the first opportunity to complete cleavage, and is stimulated by components of the backup pathway.     

Human RECQL5β stimulates flap endonuclease 1

Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5β, with the human flap endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5β dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5β and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4’s stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes. This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.     

Analysis of human flap endonuclease 1 mutants reveals a mechanism to prevent triplet repeat expansion

Flap endonuclease 1 (FEN1), involved in the joining of Okazaki fragments, has been proposed to restrain DNA repeat sequence expansion, a process associated with aging and disease. Here we analyze properties of human FEN1 having mutations at two conserved glycines (G66S and G242D) causing defects in nuclease activity. Introduction of these mutants into yeast led to sequence expansions. Reconstituting triplet repeat expansion in vitro, we previously found that DNA ligase I promotes expansion, but FEN1 prevents the ligation that forms expanded products. Here we show that among the intermediates that could generate sequence expansion, a bubble is necessary for ligation to produce the expansion product. Severe exonuclease defects in the mutant FEN1 suggested that the inability to degrade bubbles exonucleolytically leads to expansion. However, even wild type FEN1 exonuclease cannot compete with DNA ligase I to degrade a bubble structure before it can be ligated. Instead, we propose that FEN1 suppresses sequence expansion by degrading flaps that equilibrate with bubbles, thereby reducing bubble concentration. In this way FEN1 employs endonuclease rather than exonuclease to prevent expansions. A model is presented describing the roles of DNA structure, DNA ligase I, and FEN1 in sequence expansion.     

Analysis of helicase activity and substrate specificity of Drosophila RECQ5

RecQ5 is one of five RecQ helicase homologs identified in humans. Three of the human RecQ homologs (BLM, WRN and RTS) have been linked to autosomal recessive human genetic disorders (Bloom syndrome, Werner syndrome and Rothmund–Thomson syndrome, respectively) that display increased genomic instability and cause elevated levels of cancers in addition to other symptoms. To understand the role of RecQ helicases in maintaining genomic stability, the WRN, BLM and Escherichia coli RecQ helicases have been characterized in terms of their DNA substrate specificity. However, little is known about other members of the RecQ family. Here we show that Drosophila RECQ5 helicase is a structure-specific DNA helicase like the other RecQ helicases biochemically characterized so far, although the substrate specificity is not identical to that of WRN and BLM helicases. Drosophila RECQ5 helicase is capable of unwinding 3′ Flap, three-way junction, fork and three-strand junction substrates at lower protein concentrations compared to 5′ Flap, 12 nt bubble and synthetic Holliday junction structures, which can be unwound efficiently by WRN and BLM.     

Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase

Background  

Bloom syndrome is one of the most cancer-predisposing disorders and is characterized by genomic instability and a high frequency of sister chromatid exchange. The disorder is caused by loss of function of a 3' to 5' RecQ DNA helicase, BLM. The exact role of BLM in maintaining genomic integrity is not known but the helicase has been found to associate with several DNA repair complexes and some DNA replication foci.     

Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal

Eukaryotic Okazaki fragment maturation requires complete removal of the initiating RNA primer before ligation occurs. Polymerase delta (Pol delta) 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 delta 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.     

Concerted action of exonuclease and Gap-dependent endonuclease activities of FEN-1 contributes to the resolution of triplet repeat sequences (CTG)n- and (GAA)n-derived secondary structures formed during maturation of Okazaki fragments

There is much evidence to indicate that FEN-1 efficiently cleaves single-stranded DNA flaps but is unable to process double-stranded flaps or flaps adopting secondary structures. However, the absence of Fen1 in yeast results in a significant increase in trinucleotide repeat (TNR) expansion. There are then two possibilities. One is that TNRs do not always form stable secondary structures or that FEN-1 has an alternative approach to resolve the secondary structures. In the present study, we test the hypothesis that concerted action of exonuclease and gap-dependent endonuclease activities of FEN-1 play a role in the resolution of secondary structures formed by (CTG)n and (GAA)n repeats. Employing a yeast FEN-1 mutant, E176A, which is deficient in exonuclease (EXO) and gap endonuclease (GEN) activities but retains almost all of its flap endonuclease (FEN) activity, we show severe defects in the cleavage of various TNR intermediate substrates. Precise knock-in of this point mutation causes an increase in both the expansion and fragility of a (CTG)n tract in vivo. Taken together, our biochemical and genetic analyses suggest that although FEN activity is important for single-stranded flap processing, EXO and GEN activities may contribute to the resolution of structured flaps. A model is presented to explain how the concerted action of EXO and GEN activities may contribute to resolving structured flaps, thereby preventing their expansion in the genome.     

Selective cleavage of BLM, the bloom syndrome protein, during apoptotic cell death

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RECQ-like helicase that is presumed to function in mammalian DNA replication, recombination, or repair. We show here that BLM, but not the related RECQ-like helicase WRN, is rapidly cleaved in cells undergoing apoptosis. BLM was cleaved to 47- and 110-kDa major fragments, with kinetics similar to the apoptotic cleavage of poly(A)DP-ribose polymerase. BLM cleavage was prevented by a caspase 3 inhibitor and did not occur in caspase 3-deficient cells. Moreover, recombinant BLM was cleaved to 47- and 110-kDa fragments by caspase 3, but not caspase 6, in vitro. The caspase 3 recognition sequence (412)TEVD(415) was verified by mutating aspartate 415 to glycine and showing that this mutation rendered BLM resistant to caspase 3 cleavage. Cleavage did not abolish the BLM helicase activity but abolished BLM nuclear foci and the association of BLM with condensed DNA and the insoluble matrix. The results suggest that BLM, but not WRN, is an early selected target during the execution of apoptosis.     

The Werner syndrome protein is distinguished from the Bloom syndrome protein by its capacity to tightly bind diverse DNA structures

Loss of Werner syndrome helicase-exonuclease (WRN) or of its homolog Bloom syndrome helicase (BLM) results in different inherited disorders. Whereas Werner syndrome is characterized by premature onset of aging and age-associated diseases, Bloom syndrome involves developmental abnormalities and increased predisposition to diverse malignancies. To identify biochemical differences between WRN and BLM that might contribute to the dissimilar outcomes of their loss, we compared their abilities to unwind and bind in vitro diverse DNA structures. Full-length recombinant WRN and BLM proteins expressed in and purified from Sf9 insect cells unwound to comparable extents and with similar K(m) values partial DNA duplex, splayed arm DNA and G'2 bimolecular quadruplex DNA. However, WRN resolved bubble DNA ～25-fold more efficiently than BLM. The two enzymes were mainly distinguished by their contrasting abilities to bind DNA. WRN bound partial duplexes, bubble and splayed arm DNA and G'2 bimolecular and G4 four-molecular quadruplexes with dissociation constants of 0.25 to 25 nM. By contrast, BLM formed substantial complexes with only G4 quadruplex DNA while binding only marginally other DNA structures. We raise the possibility that in addition to its enzymatic activities WRN may act as a scaffold for the assembly on DNA of additional DNA processing proteins.     

洛美沙星对Bloom综合征解旋酶生物学特性影响的机理研究

Bloom综合征解旋酶(BLM)是RecQ家族DNA解旋酶中的一个重要成员,参与了DNA复制、修复、转录、重组以及端粒的维持等细胞代谢过程,在维持染色体的稳定性中具有重要作用.BLM解旋酶的突变可导致Bloom综合征.Bloom综合征是一种罕见隐性常染色体遗传疾病,患者遗传不稳定,并易患多种类型癌症.洛美沙星(LMX)可以抑制细胞内多种酶,并通过结合DNA干扰DNA代谢,从而治疗多种疾病,但是其具体的作用机理还未完全清楚.运用荧光偏振技术和自由磷检测技术,研究了LMX对BLM642～1290解旋酶DNA结合活性、解链活性和ATP酶活性的影响.运用荧光及紫外吸收光谱法研究了LMX与解旋酶结合的结合常数、结合位点数、作用力类型、结合距离等参数.结果表明,LMX与解旋酶之间能自发进行反应,两种分子有一个结合位点,通过静电引力和疏水作用力形成稳定的BLM-LMX复合物,且解旋酶的内源荧光被LMX静态猝灭,主要原因是非辐射能量转移.在这一过程中,LMX能抑制解旋酶的解链活性和ATP酶活性,而促进解旋酶的DNA结合活性.LMX对BLM解旋酶生物学活性影响的机理可能是LMX使解旋酶通过别构机制影响其ATP酶活性,并使酶的构象维持在较低解链活性的状态,通过抑制ATP催化水解与解链过程的偶联和阻止解旋酶的易位,从而抑制其解链.LMX能够促进解旋酶的DNA结合活性,可能是因为其C-6和C-7上的取代功能基团可以增加酶活力,以及增强药物、酶和DNA的结合,从而形成药物-酶-DNA复合物.这些结果为研究以DNA解旋酶为药物靶标的分子机理和理解喹诺酮类药物的作用机理奠定相关理论基础.     

Structural and functional analyses of disease-causing missense mutations in Bloom syndrome protein

Bloom syndrome (BS) is an autosomal recessive disorder characterized by genomic instability and the early development of many types of cancer. Missense mutations have been identified in the BLM gene (encoding a RecQ helicase) in affected individuals, but the molecular mechanism and the structural basis of the effects of these mutations remain to be elucidated. We analysed five disease-causing missense mutations that are localized in the BLM helicase core region: Q672R, I841T, C878R, G891E and C901Y. The disease-causing mutants had low ATPase and helicase activities but their ATP binding abilities were normal, except for Q672, whose ATP binding activity was lower than that of the intact BLM helicase. Mutants C878R, mapping near motif IV, and G891E and C901Y, mapping in motif IV, displayed severe DNA-binding defects. We used molecular modelling to analyse these mutations. Our work provides insights into the molecular basis of BLM pathology, and reveals structural elements implicated in coupling DNA binding to ATP hydrolysis and DNA unwinding. Our findings will help to explain the mechanism underlying BLM catalysis and interpreting new BLM causing mutations identified in the future.     

Multimeric BLM is dissociated upon ATP hydrolysis and functions as monomers in resolving DNA structures

Bloom (BLM) syndrome is an autosomal recessive disorder characterized by an increased risk for many types of cancers. Previous studies have shown that BLM protein forms a hexameric ring structure, but its oligomeric form in DNA unwinding is still not well clarified. In this work, we have used dynamic light scattering and various stopped-flow assays to study the active form and kinetic mechanism of BLM in DNA unwinding. It was found that BLM multimers were dissociated upon ATP hydrolysis. Steady-state and single-turnover kinetic studies revealed that BLM helicase always unwound duplex DNA in the monomeric form under conditions of varying enzyme and ATP concentrations as well as 3′-ssDNA tail lengths, with no sign of oligomerization being discerned. Measurements of ATPase activity further indicated that BLM helicase might still function as monomers in resolving highly structured DNAs such as Holliday junctions and D-loops. These results shed new light on the underlying mechanism of BLM-mediated DNA unwinding and on the molecular and functional basis for the phenotype of heterozygous carriers of BLM syndrome.     

Protein kinase A subunit expression is altered in Bloom syndrome fibroblasts and the BLM protein is increased in adrenocortical hyperplasias: inverse findings for BLM and PRKAR1A.

Bloom syndrome is a genetic disorder associated with chromosomal instability and a predisposition to tumors that is caused by germline mutations of the BLM gene, a RecQ helicase. Benign adrenocortical tumors display a degree of chromosomal instability that is more significant than benign tumors of other tissues. Cortisol-producing hyperplasias, such as primary pigmented nodular adrenocortical disease (PPNAD), which has been associated with protein kinase A (PKA) abnormalities and/or PRKAR1A mutations, also show genomic instability. Another RecQ helicase, WRN, directly interacts with the PRKAR1B subunit of PKA. In this study, we have investigated the PRKAR1A expression in primary human Bloom syndrome cell lines with known BLM mutations and examined the BLM gene expression in PPNAD and other adrenal tumor tissues. PRKAR1A and other protein kinase A (PKA) subunits were expressed in Bloom syndrome cells and their level of expression differed by subunit and cell type. Overall, fibroblasts exhibited a significant decrease in protein expression of all PKA subunits except for PRKAR1A, a pattern that has been associated with neoplastic transformation in several cell types. The BLM protein was upregulated in PPNAD and other hyperplasias, compared to samples from normal adrenals and normal cortex, as well as samples from cortisol- and aldosterone-producing adenomas (in which BLM was largely absent). These data reveal an inverse relationship between BLM and PRKAR1A: BLM deficiency is associated with a relative excess of PRKAR1A in fibroblasts compared to other PKA subunits; and PRKAR1A deficiency is associated with increased BLM protein in adrenal hyperplasias.     

RecQ-core of BLM unfolds telomeric G-quadruplex in the absence of ATP

Various helicases and single-stranded DNA (ssDNA) binding proteins are known to destabilize G-quadruplex (GQ) structures, which otherwise result in genomic instability. Bulk biochemical studies have shown that Bloom helicase (BLM) unfolds both intermolecular and intramolecular GQ in the presence of ATP. Using single molecule FRET, we show that binding of RecQ-core of BLM (will be referred to as BLM) to ssDNA in the vicinity of an intramolecular GQ leads to destabilization and unfolding of the GQ in the absence of ATP. We show that the efficiency of BLM-mediated GQ unfolding correlates with the binding stability of BLM to ssDNA overhang, as modulated by the nucleotide state, ionic conditions, overhang length and overhang directionality. In particular, we observed enhanced GQ unfolding by BLM in the presence of non-hydrolysable ATP analogs, which has implications for the underlying mechanism. We also show that increasing GQ stability, via shorter loops or higher ionic strength, reduces BLM-mediated GQ unfolding. Finally, we show that while WRN has similar activity as BLM, RecQ and RECQ5 helicases do not unfold GQ in the absence of ATP at physiological ionic strength. In summary, our study points to a novel and potentially very common mechanism of GQ destabilization mediated by proteins binding to the vicinity of these structures.     

POT1 stimulates RecQ helicases WRN and BLM to unwind telomeric DNA substrates

Defects in human RecQ helicases WRN and BLM are responsible for the cancer-prone disorders Werner syndrome and Bloom syndrome. Cellular phenotypes of Werner syndrome and Bloom syndrome, including genomic instability and premature senescence, are consistent with telomere dysfunction. RecQ helicases are proposed to function in dissociating alternative DNA structures during recombination and/or replication at telomeric ends. Here we report that the telomeric single-strand DNA-binding protein, POT1, strongly stimulates WRN and BLM to unwind long telomeric forked duplexes and D-loop structures that are otherwise poor substrates for these helicases. This stimulation is dependent on the presence of telomeric sequence in the duplex regions of the substrates. In contrast, POT1 failed to stimulate a bacterial 3'-5'-helicase. We find that purified POT1 binds to WRN and BLM in vitro and that full-length POT1 (splice variant 1) precipitates a higher amount of endogenous WRN protein, compared with BLM, from the HeLa nuclear extract. We propose roles for the cooperation of POT1 with RecQ helicases WRN and BLM in resolving DNA structures at telomeric ends, in a manner that protects the telomeric 3' tail as it is exposed during unwinding.     

The flexible loop of human FEN1 endonuclease is required for flap cleavage during DNA replication and repair

The conserved, structure-specific flap endonuclease FEN1 cleaves 5' DNA flaps that arise during replication or repair. To address in vivo mechanisms of flap cleavage, we developed a screen for human FEN1 mutants that are toxic when expressed in yeast. Two targets were revealed: the flexible loop domain and the catalytic site. Toxic mutants caused G(2) arrest and cell death and were unable to repair methyl methanesulfonate lesions. All the mutant proteins retained flap binding. Unlike the catalytic site mutants, which lacked cleavage of any 5' flaps, the loop mutants exhibited partial ability to cut 5' flaps when an adjacent single nucleotide 3' flap was present. We suggest that the flexible loop is important for efficient cleavage through positioning the 5' flap and the catalytic site.     

Structural basis of Bloom syndrome (BS) causing mutations in the BLM helicase domain

BACKGROUND: Bloom syndrome (BS) is characterized by mutations within the BLM gene. The Bloom syndrome protein (BLM) has similarity to the RecQ subfamily of DNA helicases, which contain seven conserved helicase domains and share significant sequence and structural similarity with the Rep and PcrA DNA helicases. We modeled the three-dimensional structure of the BLM helicase domain to analyze the structural basis of BS-causing mutations. MATERIALS AND METHODS: The sequence alignment was performed for RecQ DNA helicases and Rep and PcrA helicases. The crystal structure of PcrA helicase (PDB entry 3PJR) was used as the template for modeling the BLM helicase domain. The model was used to infer the function of BLM and to analyze the effect of the mutations. RESULTS: The structural model with good stereochemistry of the BLM helicase domain contains two subdomains, 1A and 2A. The electrostatic potential of the model is highly negative over most of the surface, except for the cleft between subdomains 1A and 2A which is similar to the template protein. The ATP-binding site is located inside the model between subdomains 1A and 2A; whereas, the DNA-binding region is situated at the surface cleft, with positive potential between 1A and 2A. CONCLUSIONS: The three-dimensional structure of the BLM helicase domain was modeled and applied to interpret BS-causing mutations. The mutation I841T is likely to weaken DNA binding, while the mutations C891R, C901Y, and Q672R presumably disturb the ATP binding. In addition, other critical positions are discussed.