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 IIIα. Here, we show that BLM stimulates the ability of topoisomerase IIIα to relax negatively supercoiled DNA. Moreover, DNA binding analyses indicate that BLM recruits topoisomerase IIIα to its DNA substrate. Consistent with this, a mutant form of BLM that retains helicase activity, but is unable to bind topoisomerase IIIα, fails to stimulate topoisomerase activity. These results indicate that a physical association between BLM and topoisomerase IIIα is a prerequisite for their functional biochemical interaction.     

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.     

A Novel Function of CRL4Cdt2: REGULATION OF THE SUBUNIT STRUCTURE OF DNA POLYMERASE δ IN RESPONSE TO DNA DAMAGE AND DURING THE S PHASE*

DNA polymerase δ (Pol δ4) is a heterotetrameric enzyme, whose p12 subunit is degraded in response to DNA damage, leaving behind a trimer (Pol δ3) with altered enzymatic characteristics that participate in gap filling during DNA repair. We demonstrate that CRL4^Cdt2, a key regulator of cell cycle progression that targets replication licensing factors, also targets the p12 subunit of Pol δ4 in response to DNA damage and on entry into S phase. Evidence for the involvement of CRL4^Cdt2 included demonstration that p12 possesses a proliferating cell nuclear antigen-interacting protein-degron (PIP-degron) and that knockdown of the components of the CRL4^Cdt2 complex inhibited the degradation of p12 in response to DNA damage. Analysis of p12 levels in synchronized cell populations showed that p12 is partially degraded in S phase and that this is affected by knockdowns of CUL4A or CUL4B. Laser scanning cytometry of overexpressed wild type p12 and a mutant resistant to degradation showed that the reduction in p12 levels during S phase was prevented by mutation of p12. Thus, CRL4^Cdt2 also regulates the subunit composition of Pol δ during the cell cycle. These studies reveal a novel function of CRL4^Cdt2, i.e. the direct regulation of DNA polymerase δ, adding to its known functions in the regulation of the licensing of replication origins and expanding the scope of its overall control of DNA replication. The formation of Pol δ3 in S phase as a normal aspect of cell cycle progression leads to the novel implications that it is involved in DNA replication as well as DNA repair.     

Possible anti-recombinogenic role of Bloom's syndrome helicase in double-strand break processing

Bloom’s syndrome (BS) which associates genetic instability and predisposition to cancer is caused by mutations in the BLM gene encoding a RecQ family 3′–5′ DNA helicase. It has been proposed that the generation of genetic instability in BS cells could result from an aberrant non-homologous DNA end joining (NHEJ), one of the two main DNA double-strand break (DSB) repair pathways in mammalian cells, the second major pathway being homologous recombination (HR). Using cell extracts, we report first that Ku70/80 and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), key factors of the end-joining machinery, and BLM are located in close proximity on DNA and that BLM binds to DNA only in the absence of ATP. In the presence of ATP, BLM is phosphorylated and dissociates from DNA in a strictly DNA-PKcs-dependent manner. We also show that BS cells display, in vivo, an accurate joining of DSBs, reflecting thus a functional NHEJ pathway. In sharp contrast, a 5-fold increase of the HR-mediated DNA DSB repair in BS cells was observed. These results support a model in which NHEJ activation mediates BLM dissociation from DNA, whereas, under conditions where HR is favored, e.g. at the replication fork, BLM exhibits an anti-recombinogenic role.     

Proteolysis of the Human DNA Polymerase Delta Smallest Subunit p12 by μ-Calpain in Calcium-Triggered Apoptotic HeLa Cells

Degradation of p12 subunit of human DNA polymerase delta (Pol δ) that results in an interconversion between Pol δ4 and Pol δ3 forms plays a significant role in response to replication stress or genotoxic agents triggered DNA damage. Also, the p12 is readily degraded by human calpain in vitro. However, little has been done for the investigation of its degree of participation in any of the more common apoptosis. Here, we first report that the p12 subunit is a substrate of μ-calpain. In calcium-triggered apoptotic HeLa cells, the p12 is degraded at 12 hours post-induction (hpi), restored thereafter by 24 hpi, and then depleted again after 36 hpi in a time-dependent manner while the other three subunits are not affected. It suggests a dual function of Pol δ by its interconversion between Pol δ4 and Pol δ3 that is involved in a novel unknown apoptosis mechanism. The proteolysis of p12 could be efficiently blocked by both calpain inhibitor ALLN and proteasome inhibitor MG132. In vitro pull down and co-immunoprecipitation assays show that the μ-calpain binds to p12 through the interaction of μ-calpain with Pol δ other three subunits, not p12 itself, and PCNA, implying that the proteolysis of p12 by μ-calpain might be through a Pol δ4/PCNA complex. The p12 cleavage sites by μ-calpain are further determined as the location within a 16-amino acids peptide 28-43 by in vitro cleavage assays. Thus, the p12/Pol δ is a target as a nuclear substrate of μ-calpain in a calcium-triggered apoptosis and appears to be a potential marker in the study of the chemotherapy of cancer therapies.     

Multifaceted role of the Topo IIIα–RMI1-RMI2 complex and DNA2 in the BLM-dependent pathway of DNA break end resection

BLM, a RecQ family DNA helicase mutated in Bloom''s Syndrome, participates in homologous recombination at two stages: 5′ DNA end resection and double Holliday junction dissolution. BLM exists in a complex with Topo IIIα, RMI1 and RMI2. Herein, we address the role of Topo IIIα and RMI1-RMI2 in resection using a reconstituted system with purified human proteins. We show that Topo IIIα stimulates DNA unwinding by BLM in a manner that is potentiated by RMI1-RMI2, and that the processivity of resection is reliant on the Topo IIIα–RMI1-RMI2 complex. Topo IIIα localizes to the ends of double-strand breaks, thus implicating it in the recruitment of resection factors. While the single-stranded DNA binding protein RPA plays a major role in imposing the 5′ to 3′ polarity of resection, Topo IIIα also makes a contribution in this regard. Moreover, we show that DNA2 stimulates the helicase activity of BLM. Our results thus uncover a multifaceted role of the Topo IIIα–RMI1-RMI2 ensemble and of DNA2 in the DNA resection reaction.     

Human RECQ5beta helicase promotes strand exchange on synthetic DNA structures resembling a stalled replication fork

The role of the human RECQ5β helicase in the maintenance of genomic stability remains elusive. Here we show that RECQ5β promotes strand exchange between arms of synthetic forked DNA structures resembling a stalled replication fork in a reaction dependent on ATP hydrolysis. BLM and WRN can also promote strand exchange on these structures. However, in the presence of human replication protein A (hRPA), the action of these RecQ-type helicases is strongly biased towards unwinding of the parental duplex, an effect not seen with RECQ5β. A domain within the non-conserved portion of RECQ5β is identified as being important for its ability to unwind the lagging-strand arm and to promote strand exchange on hRPA-coated forked structures. We also show that RECQ5β associates with DNA replication factories in S phase nuclei and persists at the sites of stalled replication forks after exposure of cells to UV irradiation. Moreover, RECQ5β is found to physically interact with the polymerase processivity factor proliferating cell nuclear antigen in vitro and in vivo. Collectively, these findings suggest that RECQ5β may promote regression of stalled replication forks to facilitate the bypass of replication-blocking lesions by template-switching. Loss of such activity could explain the elevated level of mitotic crossovers observed in RECQ5β-deficient cells.     

The accessory subunit B of DNA polymerase gamma is required for mitochondrial replisome function

The mitochondrial replication machinery in human cells includes the DNA polymerase γ holoenzyme and the TWINKLE helicase. Together, these two factors form a processive replication machinery, a replisome, which can use duplex DNA as template to synthesize long stretches of single-stranded DNA. We here address the importance of the smaller, accessory B subunit of DNA polymerase γ and demonstrate that this subunit is absolutely required for replisome function. The duplex DNA binding activity of the B subunit is needed for coordination of POLγ holoenzyme and TWINKLE helicase activities at the mtDNA replication fork. In the absence of proof for direct physical interactions between the components of the mitochondrial replisome, these functional interactions may explain the strict interdependence of TWINKLE and DNA polymerase γ for mitochondrial DNA synthesis. Furthermore, mutations in TWINKLE as well as in the catalytic A and accessory B subunits of the POLγ holoenzyme, may cause autosomal dominant progressive external ophthalmoplegia, a disorder associated with deletions in mitochondrial DNA. The crucial importance of the B subunit for replisome function may help to explain why mutations in these three proteins cause an identical syndrome.     

Contributions of the individual hydrophobic clefts of the Escherichia coli β sliding clamp to clamp loading, DNA replication and clamp recycling

The homodimeric Escherichia coli β sliding clamp contains two hydrophobic clefts with which proteins involved in DNA replication, repair and damage tolerance interact. Deletion of the C-terminal five residues of β (β^C) disrupted both clefts, severely impairing interactions of the clamp with the DnaX clamp loader, as well as the replicative DNA polymerase, Pol III. In order to determine whether both clefts were required for loading clamp onto DNA, stimulation of Pol III replication and removal of clamp from DNA after replication was complete, we developed a method for purification of heterodimeric clamp proteins comprised of one wild-type subunit (β⁺), and one β^C subunit (β⁺/β^C). The β⁺/β^C heterodimer interacted normally with the DnaX clamp loader, and was loaded onto DNA slightly more efficiently than was β⁺. Moreover, β⁺/β^C interacted normally with Pol III, and stimulated replication to the same extent as did β⁺. Finally, β⁺/β^C was severely impaired for unloading from DNA using either DnaX or the δ subunit of DnaX. Taken together, these findings indicate that a single cleft in the β clamp is sufficient for both loading and stimulation of Pol III replication, but both clefts are required for unloading clamp from DNA after replication is completed.     

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit

In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180_C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180_C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180_C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180_C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Ų. Importantly, in the structure of the p180_C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180_C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes.     

DNA2 Cooperates with the WRN and BLM RecQ Helicases to Mediate Long-range DNA End Resection in Human Cells

The 5′-3′ resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5′-3′ DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIIIα-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells.     

Establishing the human rolling circle reaction

Comment on: Kang YH, et al. Proc Natl Acad Sci USA 2012; 109:6042-7.In eukaryotes, the complex comprised of Mcm2–7, Cdc45 and GINS (CMG) is essential for DNA replication. Several lines of evidence indicate that the Mcm2–7 complex is the motor of the replicative helicase (reviewed in ref. ¹), which is activated by its association with Cdc45 and GINS.² Recently, we described the isolation and characterization of the human (h) CMG complex.³ In HeLa cells, this complex was formed only on chromatin and, following its isolation from cells, exhibited DNA helicase activity. Purified from Sf9 cells, hCMG possesses 3′→5′ DNA helicase activity, indicating that it moves ahead of the leading-strand DNA polymerase (pol). In contrast, the prokaryotic helicase DnaB, which unwinds DNA in the 5′→3′ direction, moves on the lagging strand. Detailed information about the progression of the prokaryotic replication fork was obtained using the rolling-circle method (ref. ⁴ and references therein). These studies permitted a detailed characterization of the joint action of the replicative pol and replicative helicase. In the rolling-circle reaction, the pol extends the 3′ end of a primer annealed to a minicircle that is then unwound simultaneously by the helicase (for a possible arrangement of proteins at the replication fork, see Fig. 1). The emerging single-stranded 5′-tail provides the template for lagging-strand synthesis. In most experiments, minicircles were engineered to contain only three nucleotides, allowing the distinction between leading- and lagging-strand nucleotide incorporation.Open in a separate windowFigure 1. Model of the human replication fork. The CMG complex unwinds DNA in the 3′→5′ direction. Polα/primase synthesizes primers to initiate leading- and lagging-strand synthesis. Polε and polδ are assigned as leading- and lagging-strand polymerases based on evidence in yeast.^5,6 Both pols require the processivity factor PCNA. RPA binds to single-stranded DNA. Additional proteins are required for DNA replication, of which only Ctf4 and Mcm10 are shown for simplicity.We initiated experiments to develop a eukaryotic replication fork in order to investigate whether the hCMG helicase activity could be coupled with the replicative pols.³ We set up rolling circle reactions using a 200-nt minicircle, the putative leading strand pol ε⁵ and hCMG and showed that DNA chains longer than 10 kb were produced (representing > 50 turns of the circle). The putative lagging strand pol δ,⁶ however, did not replace hpol ε in this reaction, though both pols extended primers on single-stranded M13 to full-length products (about 7 kb). It is tempting to speculate that an interaction between hCMG and hpol ε, but not hpol δ, contributes to their different activities. Specific interaction between GINS, a component of the CMG complex, and hpol ε has been demonstrated.⁷ However, it is presently unclear whether this contributes to the observed preferential role of pol ε and thus requires further examination.The processivity of the CMG complex alone was about 500 bp, which was stimulated to about 1 kbp by the addition of a single-strand DNA binding protein, either E. coli SSB or hRPA. The rolling circle reaction is also dependent on E. coli SSB, presumably to sequester the emerging single-stranded 5′ tail. Surprisingly, hRPA did not replace E. coli SSB in the rolling circle reaction. This was attributed to its inhibitory effects on pol ε activity in vitro. The influence of hRPA on eukaryotic fork progression is presently unclear. In the in vitro SV40 viral DNA replication system, hRPA is essential for DNA synthesis and cannot be replaced by E. coli SSB (reviewed in ref. ⁸). In this system, the SV40 large T-antigen acts as the replicative helicase, and hRPA is essential for its interaction with the hpolα/primase complex, which positions primase to initiate RNA chains. In the SV40 replication reaction, hpol δ synthesizes both leading and lagging strands. Surprisingly, while prokaryotic pols (and their processivity factors) can replace hpol δ and its auxiliary proteins in the in vitro SV40 elongation reaction, hpol ε does not play a role,⁸ suggesting that, in this system, the action of hpol ε is preferentially excluded. Importantly, no rolling circle synthesis was detected when hpol δ was used in lieu of hpol ε.³ Whether a similar mechanism leading to the exclusion of hpol δ from leading-strand synthesis is operational with the CMG helicase remains to be investigated. Using an archaeal system consisting of Pol B, RFC, PCNA, the 3′→5′ DNA helicase Mcm and the DNA primase, we have performed both leading- and lagging-strand synthesis on a rolling circle substrate.⁹ Currently, our efforts are focused on the synthesis of the lagging-strand with human proteins.In cells, the replication machinery duplicates chromatinized DNA. Thus, it is likely that chromatin remodeling factors and nucleosome chaperones play roles in the progression of the replication fork. In support of this notion, FACT was identified as a component of the yeast replisome progression complex.¹⁰ Various other proteins associate with the replication fork, such as Mcm10, Ctf4, Tim-Tipin and Claspin. The effects of these proteins on the in vitro replication reaction in eukaryotes remain to be examined.     

High-temperature single-molecule kinetic analysis of thermophilic archaeal MCM helicases

The minichromosome maintenance (MCM) complex is the replicative helicase responsible for unwinding DNA during archaeal and eukaryal genome replication. To mimic long helicase events in the cell, a high-temperature single-molecule assay was designed to quantitatively measure long-range DNA unwinding of individual DNA helicases from the archaeons Methanothermobacter thermautotrophicus (Mth) and Thermococcus sp. 9°N (9°N). Mth encodes a single MCM homolog while 9°N encodes three helicases. 9°N MCM3, the proposed replicative helicase, unwinds DNA at a faster rate compared to 9°N MCM2 and to Mth MCM. However, all three MCM proteins have similar processivities. The implications of these observations for DNA replication in archaea and the differences and similarities among helicases from different microorganisms are discussed. Development of the high-temperature single-molecule assay establishes a system to comprehensively study thermophilic replisomes and evolutionary links between archaeal, eukaryal, and bacterial replication systems.     

DNA structure and the Werner protein modulate human DNA polymerase delta-dependent replication dynamics within the common fragile site FRA16D

Common fragile sites (CFS) are chromosomal regions that exhibit instability during DNA replication stress. Although the mechanism of CFS expression has not been fully elucidated, one known feature is a severely delayed S-phase. We used an in vitro primer extension assay to examine the progression of DNA synthesis through various sequences within FRA16D by the replicative human DNA polymerases δ and α, and with human cell-free extracts. We found that specific cis-acting sequence elements perturb DNA elongation, causing inconsistent DNA synthesis rates between regions on the same strand and complementary strands. Pol δ was significantly inhibited in regions containing hairpins and microsatellites, [AT/TA]₂₄ and [A/T]_19–28, compared with a control region with minimal secondary structure. Pol δ processivity was enhanced by full length Werner Syndrome protein (WRN) and by WRN fragments containing either the helicase domain or DNA-binding C-terminal domain. In cell-free extracts, stalling was eliminated at smaller hairpins, but persisted in larger hairpins and microsatellites. Our data support a model whereby CFS expression during cellular stress is due to a combination of factors—density of specific DNA secondary-structures within a genomic region and asymmetric rates of strand synthesis.     

Telomerase-associated Protein 1, HSP90, and Topoisomerase II�� Associate Directly with the BLM Helicase in Immortalized Cells Using ALT and Modulate Its Helicase Activity Using Telomeric DNA Substrates

The BLM helicase associates with the telomere structural proteins TRF1 and TRF2 in immortalized cells using the alternative lengthening of telomere (ALT) pathways. This work focuses on identifying protein partners of BLM in cells using ALT. Mass spectrometry and immunoprecipitation techniques have identified three proteins that bind directly to BLM and TRF2 in ALT cells: telomerase-associated protein 1 (TEP1), heat shock protein 90 (HSP90), and topoisomerase IIα (TOPOIIα). BLM predominantly co-localizes with these proteins in foci actively synthesizing DNA during late S and G₂/M phases of the cell cycle when ALT is thought to occur. Immunoprecipitation studies also indicate that only HSP90 and TOPOIIα are components of a specific complex containing BLM, TRF1, and TRF2 but that this complex does not include TEP1. TEP1, TOPOIIα, and HSP90 interact directly with BLM in vitro and modulate its helicase activity on telomere-like DNA substrates but not on non-telomeric substrates. Initial studies suggest that knockdown of BLM in ALT cells reduces average telomere length but does not do so in cells using telomerase.Bloom syndrome (BS)⁴ is a genetic disease caused by mutation of both copies of the human BLM gene. It is characterized by sun sensitivity, small stature, immunodeficiency, male infertility, and an increased susceptibility to cancer of all sites and types. The high incidence of spontaneous chromosome breakage and other unique chromosomal anomalies in cells from BS patients indicate an increase in homologous recombination in somatic cells (1). Another notable feature of non-immortalized and immortalized cells from BS individuals is the presence of telomeric associations (TAs) between homologous chromosomes (2). Work from our group and others have suggested a role for BLM in recombination-mediated mechanisms of telomere elongation or ALT (alternative lengthening of telomeres), processes that maintain/elongate telomeres in the absence of telomerase (3–5). However, the exact mechanism by which BLM contributes to telomere stability is unknown.Several proteins interact with and regulate BLM helicase activity, including two telomere-specific proteins, TRF1 and TRF2 (6, 7). Although TRF2 stimulates BLM unwinding of telomeric and non-telomeric 3′-overhang substrates, TRF1 inhibits BLM unwinding of telomeric substrates. TRF2-mediated stimulation of BLM helicase activity on a telomeric substrate is observed when TRF2 is present in excess or with equimolar amount of TRF1 but not when TRF1 is present in molar excess. Both proteins associate with BLM specifically in ALT cells in vivo, suggesting their involvement in the ALT pathways. In addition to TRF1 and TRF2, the telomere single-strand DNA-binding protein POT1 strongly stimulates BLM helicase activity on long telomeric forked duplexes and D-loop structures (8). Other proteins also play an important role in telomere maintenance in telomerase-negative cells, including RAD50, NBS1, and MRE11, which co-localize with TRF1 and TRF2 in specialized ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs) (9–11). Thus, we hypothesize that BLM complex formation may be essential for the ALT mechanism, and its modification may occur dynamically during the specific nucleic acid transactions required to protect the telomere in cells using the ALT pathways.This study has identified previously unknown protein partners of BLM and TRF2 in ALT cells using double immunoprecipitation and mass spectrometry (MS). These include telomerase-associated protein 1 (TEP1), heat shock protein 90 (HSP90), and topoisomerase IIα (TOPOIIα). These proteins associate with BLM and TRF2 in cells using ALT but not in cells using telomerase and directly interact with BLM in vitro. This complex of proteins localizes to sites of new DNA synthesis in vivo in ALT cells, suggesting a role in telomere maintenance. We also identified HSP90 and TOPOIIα in another ALT-specific complex consisting of BLM, TRF1, and TRF2 but not TEP1. In vitro analyses demonstrate that HSP90 inhibits BLM helicase activity using both telomeric and non-telomeric substrates, whereas TEP1 and TOPOIIα initially slow the kinetics of BLM unwinding only using telomeric substrates. These findings suggest the presence of dynamic BLM-associated ALT complexes that include previously unidentified interacting proteins. The function of TEP1 in the BLM·TRF2 complex remains unclear, although its previously described interaction with the RNA subunit of telomerase (12) suggests an interesting hypothesis of cross-talk between mechanisms of telomere elongation.     

FF483–484 motif of human Polη mediates its interaction with the POLD2 subunit of Polδ and contributes to DNA damage tolerance

Switching between replicative and translesion synthesis (TLS) DNA polymerases are crucial events for the completion of genomic DNA synthesis when the replication machinery encounters lesions in the DNA template. In eukaryotes, the translesional DNA polymerase η (Polη) plays a central role for accurate bypass of cyclobutane pyrimidine dimers, the predominant DNA lesions induced by ultraviolet irradiation. Polη deficiency is responsible for a variant form of the Xeroderma pigmentosum (XPV) syndrome, characterized by a predisposition to skin cancer. Here, we show that the FF_483–484 amino acids in the human Polη (designated F1 motif) are necessary for the interaction of this TLS polymerase with POLD2, the B subunit of the replicative DNA polymerase δ, both in vitro and in vivo. Mutating this motif impairs Polη function in the bypass of both an N-2-acetylaminofluorene adduct and a TT-CPD lesion in cellular extracts. By complementing XPV cells with different forms of Polη, we show that the F1 motif contributes to the progression of DNA synthesis and to the cell survival after UV irradiation. We propose that the integrity of the F1 motif of Polη, necessary for the Polη/POLD2 interaction, is required for the establishment of an efficient TLS complex.     

The Human Lagging Strand DNA Polymerase δ Holoenzyme Is Distributive

Polymerase δ is widely accepted as the lagging strand replicative DNA polymerase in eukaryotic cells. It forms a replication complex in the presence of replication factor C and proliferating cell nuclear antigen to perform efficient DNA synthesis in vivo. In this study, the human lagging strand holoenzyme was reconstituted in vitro. The rate of DNA synthesis of this holoenzyme, measured with a singly primed ssM13 DNA substrate, is 4.0 ± 0.4 nucleotides. Results from adenosine 5′-(3-thiotriphosphate) tetralithium salt (ATPγS) inhibition experiments revealed the nonprocessive characteristic of the human DNA polymerase (Pol δ) holoenzyme (150 bp for one binding event), consistent with data from chase experiments with catalytically inactive mutant Pol δ^AA. The ATPase activity of replication factor C was characterized and found to be stimulated ∼10-fold in the presence of both proliferating cell nuclear antigen and DNA, but the activity was not shut down by Pol δ in accord with rapid association/dissociation of the holoenzyme to/from DNA. It is noted that high concentrations of ATP inhibit the holoenzyme DNA synthesis activity, most likely due to its inhibition of the clamp loading process.     

Structure and function of the regulatory HRDC domain from human Bloom syndrome protein

The helicase and RNaseD C-terminal (HRDC) domain, conserved among members of the RecQ helicase family, regulates helicase activity by virtue of variations in its surface residues. The HRDC domain of Bloom syndrome protein (BLM) is known as a critical determinant of the dissolution function of double Holliday junctions by the BLM–Topoisomerase IIIα complex. In this study, we determined the solution structure of the human BLM HRDC domain and characterized its DNA-binding activity. The BLM HRDC domain consists of five α-helices with a hydrophobic 3₁₀-helical loop between helices 1 and 2 and an extended acidic surface comprising residues in helices 3–5. The BLM HRDC domain preferentially binds to ssDNA, though with a markedly low binding affinity (K_d ∼100 μM). NMR chemical shift perturbation studies suggested that the critical DNA-binding residues of the BLM HRDC domain are located in the hydrophobic loop and the N-terminus of helix 2. Interestingly, the isolated BLM HRDC domain had quite different DNA-binding modes between ssDNA and Holliday junctions in electrophoretic mobility shift assay experiments. Based on its surface charge separation and DNA-binding properties, we suggest that the HRDC domain of BLM may be adapted for a unique function among RecQ helicases—that of bridging protein and DNA interactions.     

The N Terminus of the Retinoblastoma Protein Inhibits DNA Replication via a Bipartite Mechanism Disrupted in Partially Penetrant Retinoblastomas

The N-terminal domain of the retinoblastoma (Rb) tumor suppressor protein (RbN) harbors in-frame exon deletions in partially penetrant hereditary retinoblastomas and is known to impair cell growth and tumorigenesis. However, how such RbN deletions contribute to Rb tumor- and growth-suppressive functions is unknown. Here we establish that RbN directly inhibits DNA replication initiation and elongation using a bipartite mechanism involving N-terminal exons lost in cancer. Specifically, Rb exon 7 is necessary and sufficient to target and inhibit the replicative CMG helicase, resulting in the accumulation of inactive CMGs on chromatin. An independent N-terminal loop domain, which forms a projection, specifically blocks DNA polymerase α (Pol-α) and Ctf4 recruitment without affecting DNA polymerases ε and δ or the CMG helicase. Individual disruption of exon 7 or the projection in RbN or Rb, as occurs in inherited cancers, partially impairs the ability of Rb/RbN to inhibit DNA replication and block G₁-to-S cell cycle transit. However, their combined loss abolishes these functions of Rb. Thus, Rb growth-suppressive functions include its ability to block replicative complexes via bipartite, independent, and additive N-terminal domains. The partial loss of replication, CMG, or Pol-α control provides a potential molecular explanation for how N-terminal Rb loss-of-function deletions contribute to the etiology of partially penetrant retinoblastomas.