Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks

Pif-1 proteins are 5′→3′ superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3′ ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity.     

Impaired TIP60-mediated H4K16 acetylation accounts for the aberrant chromatin accumulation of 53BP1 and RAP80 in Fanconi anemia pathway-deficient cells

To rescue collapsed replication forks cells utilize homologous recombination (HR)-mediated mechanisms to avoid the induction of gross chromosomal abnormalities that would be generated by non-homologous end joining (NHEJ). Using DNA interstrand crosslinks as a replication barrier, we investigated how the Fanconi anemia (FA) pathway promotes HR at stalled replication forks. FA pathway inactivation results in Fanconi anemia, which is associated with a predisposition to cancer. FANCD2 monoubiquitination and assembly in subnuclear foci appear to be involved in TIP60 relocalization to the chromatin to acetylates histone H4K16 and prevents the binding of 53BP1 to its docking site, H4K20Me2. Thus, FA pathway loss-of-function results in accumulation of 53BP1, RIF1 and RAP80 at damaged chromatin, which impair DNA resection at stalled replication fork-associated DNA breaks and impede HR. Consequently, DNA repair in FA cells proceeds through the NHEJ pathway, which is likely responsible for the accumulation of chromosome abnormalities. We demonstrate that the inhibition of NHEJ or deacetylase activity rescue HR in FA cells.     

Recruitment of human cytomegalovirus immediate-early 2 protein onto parental viral genomes in association with ND10 in live-infected cells

The human cytomegalovirus (HCMV) immediate-early 2 (IE2) transactivator has previously been shown to form intranuclear, dot-like accumulations in association with subnuclear structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. We recently observed that IE2 can form dot-like structures even after infection of PML knockdown cells, which lack genuine ND10. To further analyze the determinants of IE2 subnuclear localization, a recombinant HCMV expressing IE2 fused to the enhanced green fluorescent protein was constructed. We infected primary human fibroblasts expressing Sp100 fused to the autofluorescent protein mCherry while performing live-cell imaging experiments. These experiments revealed a very dynamic association of IE2 dots with ND10 structures during the first hours postinfection: juxtaposed structures rapidly fused to precise co-localizations, followed by segregation, and finally, the dispersal of ND10 accumulations. Furthermore, by infecting PML knockdown cells we determined that the number of IE2 accumulations was dependent on the multiplicity of infection. Since time-lapse microscopy in live-infected cells revealed that IE2 foci developed into viral replication compartments, we hypothesized that viral DNA could act as a determinant of IE2 accumulations. Direct evidence that IE2 molecules are associated with viral DNA early after HCMV infection was obtained using fluorescence in situ hybridization. Finally, a DNA-binding-deficient IE2 mutant could no longer be recruited into viral replication centers, suggesting that the association of IE2 with viral DNA is mediated by a direct DNA contact. Thus, we identified viral DNA as an important determinant of IE2 subnuclear localization, which suggests that the formation of a virus-induced nucleoprotein complex and its spatial organization is likely to be critical at the early stages of a lytic infection.     

Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system

Initiation of DNA replication is tightly controlled during the cell cycle to maintain genome integrity. In order to directly study this control we have previously established a cell-free system from human cells that initiates semi-conservative DNA replication. Template nuclei are isolated from cells synchronized in late G₁ phase by mimosine. We have now used DNA combing to investigate initiation and further progression of DNA replication forks in this human in vitro system at single molecule level. We obtained direct evidence for bidirectional initiation of divergently moving replication forks in vitro. We assessed quantitatively replication fork initiation patterns, fork movement rates and overall fork density. Individual replication forks progress at highly heterogeneous rates (304 ± 162 bp/min) and the two forks emanating from a single origin progress independently from each other. Fork progression rates also change at the single fork level, suggesting that replication fork stalling occurs. DNA combing provides a powerful approach to analyse dynamics of human DNA replication in vitro.     

DNA binding domain in the replication checkpoint protein Mrc1 of Schizosaccharomyces pombe

The replication checkpoint is activated when replication forks are obstructed by DNA lesions or protein complexes bound to DNA or when DNA synthesis is restrained by the limited availability of deoxyribonucleotides. This checkpoint preserves genome integrity by stabilizing stalled forks and delaying the onset of mitosis. In the fission yeast Schizosaccharomyces pombe, Mrc1 is a replication checkpoint adaptor protein that allows the sensor kinase Rad3-Rad26 to activate the effector kinase Cds1. In Saccharomyces cerevisiae, Mrc1 associates with replication forks and co-precipitates with the DNA replication protein Cdc45. Whether or not Mrc1 interacts directly with DNA is unknown. Here we define a approximately 150 amino acid DNA binding domain (DBD) in the N-terminal region of S. pombe Mrc1. The DBD interacts preferentially with branched DNA structures in vitro. Deletion of the DBD or point mutations that diminish its DNA binding activity render cells sensitive to the replication inhibitor hydroxyurea. These mutations also impair the replication checkpoint arrest. The DBD has a helix-loop-helix motif that is predicted to bind DNA. This motif is conserved in the recently identified N-terminal DBD of human Claspin, a presumptive homolog of yeast Mrc1 proteins.     

Cleavage of stalled forks by fission yeast Mus81/Eme1 in absence of DNA replication checkpoint

During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase-specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed.     

Identification and characterization of human MUS81-MMS4 structure-specific endonuclease

Replication forks may stall when they reach a block on the DNA template such as DNA damage, and the recovery of such stalled replication forks plays a crucial role in the maintenance of genomic stability. Holliday junctions, which are X-shaped DNA structures, are formed at the stalled replication forks and can accumulate if they are not cleaved by structure-specific endonucleases. Recently, a novel nuclease involved in resolving Holliday junction-like structures, Mus81, has been reported in yeast and humans. MUS81 has sequence homology to another DNA nuclease, XPF, which, with its partner ERCC1, makes the 5' incision during nucleotide excision repair. MUS81 also has a binding partner named Mms4 in Saccharomyces cerevisiae and Eme1 in Schizosaccharomyces pombe, but no such partner was identified in human cells. Here, we report identification of the binding partner of human MUS81, which we designate hMMS4. Using immunoaffinity purification we show that hMUS81 or hMMS4 alone have no detectable nuclease activity, but that the hMUS81.hMMS4 complex is a structure-specific nuclease that is capable of resolving fork structures.     

TopBP1 localises to centrosomes in mitosis and to chromosome cores in meiosis

Topoisomerase IIbeta binding protein 1 (TopBP1), previously shown to localise to sites of DNA damage and to stalled replication forks, has been implicated in DNA replication and in DNA damage response. In this work we showed that TopBP1 was localised in structures other than stalled replication forks. In late mitosis TopBP1 localises to centrosomes in a manner similar to other DNA damage response proteins such as BRCA1 and p53. Spindle checkpoint activation does not affect this centrosomal localisation. Moreover, in the testis, we detected high levels of TopBP1 associated with meiotic prophase chromosome cores and the X-Y pair. Together, these data suggest a direct role of TopBP1 during both mitosis and meiotic prophase I.     

Bloom's syndrome protein is required for correct relocalization of RAD50/MRE11/NBS1 complex after replication fork arrest

Bloom's syndrome (BS) is a rare genetic disorder characterized by a broad range of symptoms and, most importantly, a predisposition to many types of cancers. Cells derived from patients with BS exhibit an elevated rate of somatic recombination and hypermutability, supporting a role for bleomycin (BLM) in the maintenance of genomic integrity. BLM is thought to participate in several DNA transactions, the failure of which could give raise to genomic instability, and to interact with many proteins involved in replication, recombination, and repair. In this study, we show that BLM function is specifically required to properly relocalize the RAD50/MRE11/NBS1 (RMN) complex at sites of replication arrest, but is not essential in the activation of BRCA1 either after stalled replication forks or gamma-rays. We also provide evidence that BLM is phosphorylated after replication arrest in an Ataxia and RAD3-related protein (ATR)-dependent manner and that phosphorylation is not required for subnuclear relocalization. Therefore, in ATR dominant negative mutant cells, the assembly of the RMN complex in nuclear foci after replication blockage is almost completely abolished. Together, these results suggest a relationship between BLM, ATR, and the RMN complex in the response to replication arrest, proposing a role for BLM protein and RMN complex in the resolution of stalled replication forks.     

The DNA repair endonuclease XPG interacts directly and functionally with the WRN helicase defective in Werner syndrome

XPG is a structure-specific endonuclease required for nucleotide excision repair (NER). XPG incision defects result in the cancer-prone syndrome xeroderma pigmentosum, whereas truncating mutations of XPG cause the severe postnatal progeroid developmental disorder Cockayne syndrome. We show that XPG interacts directly with WRN protein, which is defective in the premature aging disorder Werner syndrome, and that the two proteins undergo similar subnuclear redistribution in S phase and colocalize in nuclear foci. The co-localization was observed in mid- to late S phase, when WRN moves from nucleoli to nuclear foci that have been shown to contain both protein markers of stalled replication forks and telomeric proteins. We mapped the interaction between XPG and WRN to the C-terminal domains of each, and show that interaction with the C-terminal domain of XPG strongly stimulates WRN helicase activity. WRN also possesses a competing DNA single-strand annealing activity that, combined with unwinding, has been shown to coordinate regression of model replication forks to form Holliday junction/chicken foot intermediate structures. We tested whether XPG stimulated WRN annealing activity, and found that XPG itself has intrinsic strand annealing activity that requires the unstructured R- and C-terminal domains but not the conserved catalytic core or endonuclease activity. Annealing by XPG is cooperative, rather than additive, with WRN annealing. Taken together, our results suggest a novel function for XPG in S phase that is, at least in part, performed coordinately with WRN, and which may contribute to the severity of the phenotypes that occur upon loss of XPG.Key words: Cockayne syndrome, progeria, DNA annealing, DNA replication, DNA damage response     

Mus81-Eme1 and Rqh1 involvement in processing stalled and collapsed replication forks

The processing of stalled replication forks and the repair of collapsed replication forks are essential functions in all organisms. In fission yeast DNA junctions at stalled replication forks appear to be processed by either the Rqh1 DNA helicase or Mus81-Eme1 endonuclease. Accordingly, we show that the hypersensitivity to agents that cause replication fork stalling of mus81, eme1, and rqh1 mutants is suppressed by a Holliday junction resolvase (RusA), as is the synthetic lethality of a mus81(-) rqh1(-) double mutant. Recombinant Mus81-Eme1, purified from Escherichia coli, readily cleaves replication fork structures but cleaves synthetic Holliday junctions relatively poorly in vitro. From these data we propose that Mus81-Eme1 can process stalled replication forks before they have regressed to form a Holliday junction. We also implicate Mus81-Eme1 and Rqh1 in the repair of collapsed replication forks. Here Mus81-Eme1 and Rqh1 seem to function on different substrates because RusA can substitute for Mus81-Eme1 but not Rqh1.     

Human claspin is a ring-shaped DNA-binding protein with high affinity to branched DNA structures

Claspin is an essential protein for the ATR-dependent activation of the DNA replication checkpoint response in Xenopus and human cells. Here we describe the purification and characterization of human Claspin. The protein has a ring-like structure and binds with high affinity to branched DNA molecules. These findings suggest that Claspin may be a component of the replication ensemble and plays a role in the replication checkpoint by directly associating with replication forks and with the various branched DNA structures likely to form at stalled replication forks because of DNA damage.     

Permanent attachment of replication origins to the nuclear matrix in BHK-cells.

The position of replication origins and replication forks relative to the nuclear matrix was analysed by autoradiography. Analysis of 2M NaCl-extracted extracted BHK-nuclei, prepared on coverslips, showed that after brief pulses grains were exclusively found over the central core of the residual nuclei, which corresponds to positions in the nuclear matrix. In asynchronous cells these grains were found to migrate into the DNA-halo surrounding the matrix during a subsequent chase. When the pulse had been administered to synchronous cells at the onset of S-phase, it was observed, however, that in the majority of the structures no such migration had occurred. From this, and from the fact that label incorporated later in S-phase could be chased into the halo, we conclude that, contrary to DNA in replication forks, DNA containing replication origins is permanently attached to the nuclear matrix.     

Histone H2AX and Fanconi anemia FANCD2 function in the same pathway to maintain chromosome stability

Fanconi anemia (FA) is a chromosome fragility syndrome characterized by bone marrow failure and cancer susceptibility. The central FA protein FANCD2 is known to relocate to chromatin upon DNA damage in a poorly understood process. Here, we have induced subnuclear accumulation of DNA damage to prove that histone H2AX is a novel component of the FA/BRCA pathway in response to stalled replication forks. Analyses of cells from H2AX knockout mice or expressing a nonphosphorylable H2AX (H2AX(S136A/S139A)) indicate that phosphorylated H2AX (gammaH2AX) is required for recruiting FANCD2 to chromatin at stalled replication forks. FANCD2 binding to gammaH2AX is BRCA1-dependent and cells deficient or depleted of H2AX show an FA-like phenotype, including an excess of chromatid-type chromosomal aberrations and hypersensitivity to MMC. This MMC hypersensitivity of H2AX-deficient cells is not further increased by depleting FANCD2, indicating that H2AX and FANCD2 function in the same pathway in response to DNA damage-induced replication blockage. Consequently, histone H2AX is functionally connected to the FA/BRCA pathway to resolve stalled replication forks and prevent chromosome instability.     

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.     

Swi1 and Swi3 are components of a replication fork protection complex in fission yeast

Swi1 is required for programmed pausing of replication forks near the mat1 locus in the fission yeast Schizosaccharomyces pombe. This fork pausing is required to initiate a recombination event that switches mating type. Swi1 is also needed for the replication checkpoint that arrests division in response to fork arrest. How Swi1 accomplishes these tasks is unknown. Here we report that Swi1 copurifies with a 181-amino-acid protein encoded by swi3(+). The Swi1-Swi3 complex is required for survival of fork arrest and for activation of the replication checkpoint kinase Cds1. Association of Swi1 and Swi3 with chromatin during DNA replication correlated with movement of the replication fork. swi1Delta and swi3Delta mutants accumulated Rad22 (Rad52 homolog) DNA repair foci during replication. These foci correlated with the Rad22-dependent appearance of Holliday junction (HJ)-like structures in cells lacking Mus81-Eme1 HJ resolvase. Rhp51 and Rhp54 homologous recombination proteins were not required for viability in swi1Delta or swi3Delta cells, indicating that the HJ-like structures arise from single-strand DNA gaps or rearranged forks instead of broken forks. We propose that Swi1 and Swi3 define a fork protection complex that coordinates leading- and lagging-strand synthesis and stabilizes stalled replication forks.     

Monitoring S phase progression globally and locally using BrdU incorporation in TK(+) yeast strains

Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK(+) yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8-9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 'early' origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.     

Structural and functional relationships of FAN1

FANCD2/FANCI-associated nuclease (FAN1) is a 5′ flap structure-specific endonuclease and 5′ to 3′ exonuclease. This nuclease can resolve interstrand cross-links (ICLs) independently of the Fanconi anemia (FA) pathway and controls the progression of stalled replication forks in an FA-dependent manner, thereby maintaining chromosomal stability. Several FAN1 mutations are observed in various cancers and degenerative diseases. Recently, several crystal structures of the FAN1-DNA complexes have been reported, and to date, these represent the only structures for a DNA bound ICL-repair nuclease. Puzzlingly, human FAN1 forms two different quaternary structures with different DNA binding modes, and based on these structures, two ICL-repair mechanisms have been proposed. In one mechanism, monomeric FAN1 recognizes the 5′ flap terminal phosphate via a basic pocket and successively cleaves at every third nucleotide of the DNA substrates. In the other mechanism, dimeric FAN1 scans, latches, and unwinds the postnick duplex of the substrate DNA to direct the scissile phosphodiester group to the active site. In this review, we discuss the structures, function, and proposed mechanisms of FAN1 nuclease, and provide the insights into its role in ICL repair and in processing of stalled replication forks.     

CHK1 activity is required for continuous replication fork elongation but not stabilization of post-replicative gaps after UV irradiation

Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine-inhibiting ATM and ATR amongst other kinases-is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase η (Polη), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.     

Novel function of the flap endonuclease 1 complex in processing stalled DNA replication forks

Restarting stalled replication forks partly depends on the break-induced recombination pathway, in which a DNA double-stranded break (DSB) is created on the stalled replication fork to initiate the downstream recombination cascades. Single-stranded DNA gaps accumulating on stalled replication forks are potential targets for endonucleases to generate DSBs. However, it is unclear how this process is executed and which nucleases are involved in eukaryotic cells. Here, we identify a novel gap endonuclease (GEN) activity of human flap endonuclease 1 (FEN-1), critical in resolving stalled replication fork. In response to replication arrest, FEN-1 interacts specifically with Werner syndrome protein for efficient fork cleavage. Replication protein A facilitates FEN-1 interaction with DNA bubble structures. Human FEN-1, but not the GEN-deficient mutant, E178A, was shown to rescue the defect in resistance to UV and camptothecin in a yeast FEN-1 null mutant.