FANCD2-Controlled Chromatin Access of the Fanconi-Associated Nuclease FAN1 Is Crucial for the Recovery of Stalled Replication Forks

Fanconi anemia (FA) is a cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand cross-links (ICLs). Within the FA pathway, an upstream core complex monoubiquitinates and recruits the FANCD2 protein to ICLs on chromatin. Ensuing DNA repair involves the Fanconi-associated nuclease 1 (FAN1), which interacts selectively with monoubiquitinated FANCD2 (FANCD2^Ub) at ICLs. Importantly, FANCD2 has additional independent functions: it binds chromatin and coordinates the restart of aphidicolin (APH)-stalled replication forks in concert with the BLM helicase, while protecting forks from nucleolytic degradation by MRE11. We identified FAN1 as a new crucial replication fork recovery factor. FAN1 joins the BLM-FANCD2 complex following APH-mediated fork stalling in a manner dependent on MRE11 and FANCD2, followed by FAN1 nuclease-mediated fork restart. Surprisingly, APH-induced activation and chromatin recruitment of FAN1 occur independently of the FA core complex or the FAN1 UBZ domain, indicating that the FANCD2^Ub isoform is dispensable for functional FANCD2-FAN1 cross talk during stalled fork recovery. In the absence of FANCD2, MRE11 exonuclease-promoted access of FAN1 to stalled forks results in severe FAN1-mediated nucleolytic degradation of nascent DNA strands. Thus, FAN1 nuclease activity at stalled replication forks requires tight regulation: too little inhibits fork restart, whereas too much causes fork degradation.     

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.     

Human KIAA1018/FAN1 localizes to stalled replication forks via its ubiquitin-binding domain

Genome maintenance pathways correct aberrations in DNA that would be deleterious to the organism. A crucial element of many genome maintenance processes is the ability to degrade DNA that either contains errors or obscures useful substrates for recombination and/or repair by means of nucleases. We have examined a putative nuclease that has heretofore been unreported, KIAA1018/FAN1. This protein contains a predicted ubiquitin-binding zinc finger domain (UBZ) near its N-terminus and an endonuclease-like fold near its C-terminus. Here we describe that FAN1 is a nuclear protein and forms DNA-damage-induced foci, which appear to be at stalled replication forks as denoted by RPA colocalization. Localization of FAN1 to sites of damage is dependent upon its UBZ domain. In addition, knockdown of FAN1 by RNA interference leads to increased sensitivity to interstrand crosslinking agents and accumulation of abnormal chromosomes. FAN1 may be an important new player in the maintenance of genome stability.     

Identification and characterization of the human mus81-eme1 endonuclease

The faithful and complete replication of DNA is necessary for the maintenance of genome stability. It is known, however, that replication forks stall at lesions in the DNA template and need to be processed so that replication restart can occur. In fission yeast, the Mus81-Eme1 endonuclease complex (Mus81-Mms4 in Saccharomyces cerevisiae) has been implicated in the processing of aberrant replication intermediates. In this report, we identify the human homolog of the Schizosaccharomyces pombe EME1 gene and have purified the human Mus81-Eme1 heterodimer. We show that Mus81-Eme1 is an endonuclease that exhibits a high specificity for synthetic replication fork structures and 3'-flaps in vitro. The nuclease cleaves Holliday junctions inefficiently ( approximately 75-fold less than flap or fork structures), although cleavage can be increased 6-fold by the presence of homologous sequences previously shown to permit base pair "breathing." We conclude that human Mus81-Eme1 is a flap/fork endonuclease that is likely to play a role in the processing of stalled replication fork intermediates.     

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.     

Rad9/53BP1 protects stalled replication forks from degradation in Mec1/ATR‐defective cells

Nucleolytic processing by nucleases can be a relevant mechanism to allow repair/restart of stalled replication forks. However, nuclease action needs to be controlled to prevent overprocessing of damaged replication forks that can be detrimental to genome stability. The checkpoint protein Rad9/53BP1 is known to limit nucleolytic degradation (resection) of DNA double‐strand breaks (DSBs) in both yeast and mammals. Here, we show that loss of the inhibition that Rad9 exerts on resection exacerbates the sensitivity to replication stress of Mec1/ATR‐defective yeast cells by exposing stalled replication forks to Dna2‐dependent degradation. This Rad9 protective function is independent of checkpoint activation and relies mainly on Rad9‐Dpb11 interaction. We propose that Rad9/53BP1 supports cell viability by protecting stalled replication forks from extensive resection when the intra‐S checkpoint is not fully functional.     

Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent‐strand DNA

Degradation and collapse of stalled replication forks are main sources of genomic instability, yet the molecular mechanisms for protecting forks from degradation/collapse are not well understood. Here, we report that human CST (CTC1‐STN1‐TEN1) proteins, which form a single‐stranded DNA‐binding complex, localize at stalled forks and protect stalled forks from degradation by the MRE11 nuclease. CST deficiency increases MRE11 binding to stalled forks, leading to nascent‐strand degradation at reversed forks and ssDNA accumulation. In addition, purified CST complex binds to 5’ DNA overhangs and directly blocks MRE11 degradation in vitro, and the DNA‐binding ability of CST is required for blocking MRE11‐mediated nascent‐strand degradation. Our results suggest that CST inhibits MRE11 binding to reversed forks, thus antagonizing excessive nascent‐strand degradation. Finally, we uncover that CST complex inactivation exacerbates genome instability in BRCA2 deficient cells. Collectively, our findings identify the CST complex as an important fork protector that preserves genome integrity under replication perturbation.     

Human Fanconi Anemia Complementation Group A Protein Stimulates the 5’ Flap Endonuclease Activity of FEN1

In eukaryotic cells, Flap endonuclease 1 (FEN1) is a major structure-specific endonuclease that processes 5’ flapped structures during maturation of lagging strand DNA synthesis, long patch base excision repair, and rescue of stalled replication forks. Here we report that fanconi anemia complementation group A protein (FANCA), a protein that recognizes 5’ flap structures and is involved in DNA repair and maintenance of replication forks, constantly stimulates FEN1-mediated incision of both DNA and RNA flaps. Kinetic analyses indicate that FANCA stimulates FEN1 by increasing the turnover rate of FEN1 and altering its substrate affinity. More importantly, six pathogenic FANCA mutants are significantly less efficient than the wild-type at stimulating FEN1 endonuclease activity, implicating that regulation of FEN1 by FANCA contributes to the maintenance of genomic stability.     

Exo1 processes stalled replication forks and counteracts fork reversal in checkpoint-defective cells

The replication checkpoint coordinates the cell cycle with DNA replication and recombination, preventing genome instability and cancer. The budding yeast Rad53 checkpoint kinase stabilizes stalled forks and replisome-fork complexes, thus preventing the accumulation of ss-DNA regions and reversed forks at collapsed forks. We searched for factors involved in the processing of stalled forks in HU-treated rad53 cells. Using the neutral-neutral two-dimensional electrophoresis technique (2D gel) and psoralen crosslinking combined with electron microscopy (EM), we found that the Exo1 exonuclease is recruited to stalled forks and, in rad53 mutants, counteracts reversed fork accumulation by generating ss-DNA intermediates. Hence, Exo1-mediated fork processing resembles the action of E. coli RecJ nuclease at damaged forks. Fork stability and replication restart are influenced by both DNA polymerase-fork association and Exo1-mediated processing. We suggest that Exo1 counteracts fork reversal by resecting newly synthesized chains and resolving the sister chromatid junctions that cause regression of collapsed forks.     

A critical role of the 3' terminus of nascent DNA chains in recognition of stalled replication forks

Arrest of replication forks by various internal and external threats evokes a myriad of cellular reactions, collectively known as DNA replication checkpoint responses. In bacteria, PriA is essential for restoration of stalled replication forks and recombinational repair of double-stranded DNA breaks and is a candidate sensor protein that may recognize arrested forks. Here, we report that PriA protein specifically recognizes 3' termini of arrested nascent DNA chains at model stalled replication forks in vitro. Mutations in the putative "3' terminus binding pocket" present in the N-terminal segment of PriA result in reduced binding to stalled replication fork structures and loss of its biological functions. The results suggest a mechanism by which stalled replication forks are recognized by a sensor protein for checkpoint responses.     

Multiple but dissectible functions of FEN-1 nucleases in nucleic acid processing, genome stability and diseases

Flap EndoNuclease-1 (FEN-1) is a multifunctional and structure-specific nuclease involved in nucleic acid processing pathways. It plays a critical role in maintaining human genome stability through RNA primer removal, long-patch base excision repair and resolution of dinucleotide and trinucleotide repeat secondary structures. In addition to its flap endonuclease (FEN) and nick exonuclease (EXO) activities, a new gap endonuclease (GEN) activity has been characterized. This activity may be important in apoptotic DNA fragmentation and in resolving stalled DNA replication forks. The multiple functions of FEN-1 are regulated via several means, including formation of complexes with different protein partners, nuclear localization in response to cell cycle or DNA damage and post-translational modifications. Its functional deficiency is predicted to cause genetic diseases, including Huntington's disease, myotonic dystrophy and cancers. This review summarizes the knowledge gained through efforts in the past decade to define its structural elements for specific activities and possible pathological consequences of altered functions of this multirole player.     

Stalled replication forks: Making ends meet for recognition and stabilization

In bacteria, PriA protein, a conserved DEXH‐type DNA helicase, plays a central role in replication restart at stalled replication forks. Its unique DNA‐binding property allows it to recognize and stabilize stalled forks and the structures derived from them. Cells must cope with fork stalls caused by various replication stresses to complete replication of the entire genome. Failure of the stalled fork stabilization process and eventual restart could lead to various forms of genomic instability. The low viability of priA null cells indicates a frequent occurrence of fork stall during normal growth that needs to be properly processed. PriA specifically recognizes the 3′‐terminus of the nascent leading strand or the invading strand in a displacement (D)‐loop by the three‐prime terminus binding pocket (TT‐pocket) present in its unique DNA binding domain. Elucidation of the structural basis for recognition of arrested forks by PriA should provide useful insight into how stalled forks are recognized in eukaryotes.     

DNA2 drives processing and restart of reversed replication forks in human cells

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.     

Replication fork dynamics and the DNA damage response

Prevention and repair of DNA damage is essential for maintenance of genomic stability and cell survival. DNA replication during S-phase can be a source of DNA damage if endogenous or exogenous stresses impair the progression of replication forks. It has become increasingly clear that DNA-damage-response pathways do not only respond to the presence of damaged DNA, but also modulate DNA replication dynamics to prevent DNA damage formation during S-phase. Such observations may help explain the developmental defects or cancer predisposition caused by mutations in DNA-damage-response genes. The present review focuses on molecular mechanisms by which DNA-damage-response pathways control and promote replication dynamics in vertebrate cells. In particular, DNA damage pathways contribute to proper replication by regulating replication initiation, stabilizing transiently stalled forks, promoting replication restart and facilitating fork movement on difficult-to-replicate templates. If replication fork progression fails to be rescued, this may lead to DNA damage and genomic instability via nuclease processing of aberrant fork structures or incomplete sister chromatid separation during mitosis.     

The DNA repair component Metnase regulates Chk1 stability

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylation of downstream effectors. Metnase (also termed SETMAR) is a SET histone methylase and transposase nuclease protein that promotes both DNA double strand break (DSB) repair and re-start of stalled replication forks. We previously found that Chk1 phosphorylation of Metnase on S495 enhanced its DNA DSB repair activity but decreased its ability to re-start stalled replication forks. Here we show that phosphorylated Metnase feeds back to increase the half-life of Chk1. Chk1 half-life is regulated by DDB1 targeting it to Cul4A for ubiquitination and destruction. Metnase decreases Chk1 interaction with DDB1, and decreases Chk1 ubiquitination. These data define a novel pathway for Chk1 regulation, whereby a target of Chk1, Metnase, feeds back to amplify Chk1 stability, and therefore enhance replication fork arrest.     

Recruitment to stalled replication forks of the PriA DNA helicase and replisome-loading activities is essential for survival

PriA, a 3′ → 5′ superfamily 2 DNA helicase, acts to remodel stalled replication forks and as a specificity factor for origin-independent assembly of a new replisome at the stalled fork. The ability of PriA to initiate replication at stalled forked structures ensures complete genome replication and helps to protect the cell from illegitimate recombination events. This review focuses on the activities of PriA and its role in replication fork assembly and maintaining genomic integrity.     

EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair

Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.     

The intra-S phase checkpoint targets Dna2 to prevent stalled replication forks from reversing

When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and ～10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression-regressed leading or lagging strands-on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.     

DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1

To ensure proper replication and segregation of the genome, eukaryotic cells have evolved surveillance systems that monitor and react to impaired replication fork progression. In budding yeast, the intra-S phase checkpoint responds to stalled replication forks by downregulating late-firing origins, preventing spindle elongation and allowing efficient resumption of DNA synthesis after recovery from stress. Mutations in this pathway lead to high levels of genomic instability, particularly in the presence of DNA damage. Here we demonstrate by chromatin immunoprecipitation that when yeast replication forks stall due to hydroxyurea (HU) treatment, DNA polymerases alpha and epsilon are stabilized for 40-60 min. This requires the activities of Sgs1, a member of the RecQ family of DNA helicases, and the ATM-related kinase Mec1, but not Rad53 activation. A model is proposed whereby Sgs1 helicase resolves aberrantly paired structures at stalled forks to maintain single-stranded DNA that allows RP-A and Mec1 to promote DNA polymerase association.