Wojciech Niedzwiedz

The vertebrate Hef ortholog is a component of the Fanconi anemia tumor-suppressor pathway.

The helicase-associated endonuclease for fork-structured DNA (Hef) is an archaeabacterial protein that processes blocked replication forks. Here we have isolated the vertebrate Hef ortholog and investigated its molecular function. Disruption of this gene in chicken DT40 cells results in genomic instability and sensitivity to DNA cross-links. The similarity of this phenotype to that of cells lacking the Fanconi anemia–related (FA) tumor-suppressor genes led us to investigate whether Hef functions in this pathway. Indeed, we found a genetic interaction between the FANCC and Hef genes. In addition, Hef is a component of the FA nuclear protein complex that facilitates its DNA damage–inducible chromatin localization and the monoubiquitination of the FA protein FANCD2. Notably, Hef interacts directly with DNA structures that are intermediates in DNA replication. This discovery sheds light on the origins, regulation and molecular function of the FA tumor-suppressor pathway in the maintenance of genome stability.

ATR activation and replication fork restart are defective in FANCM‐deficient cells

Fanconi anaemia is a chromosomal instability disorder associated with cancer predisposition and bone marrow failure. Among the 13 identified FA gene products only one, the DNA translocase FANCM, has homologues in lower organisms, suggesting a conserved function in DNA metabolism. However, a precise role for FANCM in DNA repair remains elusive. Here, we show a novel function for FANCM that is distinct from its role in the FA pathway: promoting replication fork restart and simultaneously limiting the accumulation of RPA‐ssDNA. We show that in DT40 cells this process is controlled by ATR and PLK1, and that in the absence of FANCM, stalled replication forks are unable to resume DNA synthesis and genome duplication is ensured by excess origin firing. Unexpectedly, we also uncover an early role for FANCM in ATR‐mediated checkpoint signalling by promoting chromatin retention of TopBP1. Failure to retain TopBP1 on chromatin impacts on the ability of ATR to phosphorylate downstream molecular targets, including Chk1 and SMC1. Our data therefore indicate a fundamental role for FANCM in the maintenance of genome integrity during S phase.

The Fanconi Anemia Pathway Maintains Genome Stability by Coordinating Replication and Transcription.

Summary DNA replication stress can cause chromosomal instability and tumor progression. One key pathway that counteracts replication stress and promotes faithful DNA replication consists of the Fanconi anemia (FA) proteins. However, how these proteins limit replication stress remains largely elusive. Here we show that conflicts between replication and transcription activate the FA pathway. Inhibition of transcription or enzymatic degradation of transcription-associated R-loops (DNA:RNA hybrids) suppresses replication fork arrest and DNA damage occurring in the absence of a functional FA pathway. Furthermore, we show that simple aldehydes, known to cause leukemia in FA-deficient mice, induce DNA:RNA hybrids in FA-depleted cells. Finally, we demonstrate that the molecular mechanism by which the FA pathway limits R-loop accumulation requires FANCM translocase activity. Failure to activate a response to physiologically occurring DNA:RNA hybrids may critically contribute to the heightened cancer predisposition and bone marrow failure of individuals with mutated FA proteins.

The Walker B motif in avian FANCM is required to limit sister chromatid exchanges but is dispensable for DNA crosslink repair

FANCM, the most highly conserved component of the Fanconi Anaemia (FA) pathway can resolve recombination intermediates and remodel synthetic replication forks. However, it is not known if these activities are relevant to how this conserved protein activates the FA pathway and promotes DNA crosslink repair. Here we use chicken DT40 cells to systematically dissect the function of the helicase and nuclease domains of FANCM. Our studies reveal that these domains contribute distinct roles in the tolerance of crosslinker, UV light and camptothecin-induced DNA damage. Although the complete helicase domain is critical for crosslink repair, a predicted inactivating mutation of the Walker B box domain has no impact on FA pathway associated functions. However, this mutation does result in elevated sister chromatid exchanges (SCE). Furthermore, our genetic dissection indicates that FANCM functions with the Blm helicase to suppress spontaneous SCE events. Overall our results lead us to reappraise the role of helicase domain associated activities of FANCM with respect to the activation of the FA pathway, crosslink repair and in the resolution of recombination intermediates.

FANCJ couples replication past natural fork barriers with maintenance of chromatin structure

The FANCJ helicase promotes DNA replication in trans by counteracting fork stalling at replication barriers and suppresses heterochromatin spreading by coupling fork movement with maintenance of chromatin structure.

The DNA translocase activity of FANCM protects stalled replication forks

FANCM is the most highly conserved protein within the Fanconi anaemia (FA) tumour suppressor pathway. However, although FANCM contains a helicase domain with translocase activity, this is not required for its role in activating the FA pathway. Instead, we show here that FANCM translocaseactivity is essential for promoting replication fork stability. We demonstrate that cells expressing translocase-defective FANCM show altered global replication dynamics due to increased accumulation of stalled forks that subsequently degenerate into DNA double-strand breaks, leading to ATM activation, CTBP-interacting protein (CTIP)-dependent end resection and homologous recombination repair. Accordingly, abrogation of ATM or CTIP function in FANCM-deficient cells results in decreased cell survival. We also found that FANCM translocase activity protects cells from accumulating 53BP1-OPT domains, which mark lesions resulting from problems arising during replication. Taken together, these data show that FANCM plays an essential role in maintaining chromosomal integrity by promoting the recovery of stalled replication forks and hence preventing tumourigenesis.

BOD1L Is Required to Suppress Deleterious Resection of Stressed Replication Forks

Recognition and repair of damaged replication forks are essential to maintain genome stability and are coordinated by the combined action of the Fanconi anemia and homologous recombination pathways. These pathways are vital to protect stalled replication forks from uncontrolled nucleolytic activity, which otherwise causes irreparable genomic damage. Here, we identify BOD1L as a component of this fork protection pathway, which safeguards genome stability after replication stress. Loss of BOD1L confers exquisite cellular sensitivity to replication stress and uncontrolled resection of damaged replication forks, due to a failure to stabilize RAD51 at these forks. Blocking DNA2-dependent resection, or downregulation of the helicases BLM and FBH1, suppresses both catastrophic fork processing and the accumulation of chromosomal damage in BOD1L-deficient cells. Thus, our work implicates BOD1L as a critical regulator of genome integrity that restrains nucleolytic degradation of damaged replication forks.

Visualization of DNA replication in the vertebrate model system DT40 using the DNA fiber technique.

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development. Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

TOPBP1 recruits TOP2A to ultra-fine anaphase bridges to aid in their resolution

During mitosis, sister chromatids must be faithfully segregated to ensure that daughter cells receive one copy of each chromosome. However, following replication they often remain entangled. Topoisomerase IIα (TOP2A) has been proposed to resolve such entanglements, but the mechanisms governing TOP2A recruitment to these structures remain poorly understood. Here, we identify TOPBP1 as a novel interactor of TOP2A, and reveal that it is required for TOP2A recruitment to ultra-fine anaphase bridges (UFBs) in mitosis. The C-terminal region of TOPBP1 interacts with TOP2A, and TOPBP1 recruitment to UFBs requires its BRCT domain 5. Depletion of TOPBP1 leads to accumulation of UFBs, the majority of which arise from centromeric loci. Accordingly, expression of a TOPBP1 mutant that is defective in TOP2A binding phenocopies TOP2A depletion. These findings provide new mechanistic insights into how TOP2A promotes resolution of UFBs during mitosis, and highlights a pivotal role for TOPBP1 in this process.

Mutations in CDC45, Encoding an Essential Component of the Pre-initiation Complex, Cause Meier-Gorlin Syndrome and Craniosynostosis

DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis.