The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL.

The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL.

Motor-mediated cortical versus astral microtubule organization in lipid-monolayered droplets.

The correct spatial organization of microtubules is of crucial importance for determining the internal architecture of eukaryotic cells. Microtubules are arranged in space by a multitude of biochemical activities and by spatial constraints imposed by the cell boundary. The principles underlying the establishment of distinct intracellular architectures are only poorly understood.

Preparing to unwind.

A combination of protein crystallography and biochemistry has revealed how a ring-shaped helicase might trap a single DNA strand as the double helix melts, and before it is unwound.

Structure of the human FANCL RING-Ube2T complex reveals determinants of cognate E3-E2 selection.

The combination of an E2 ubiquitin-conjugating enzyme with an E3 ubiquitin-ligase is essential for ubiquitin modification of a substrate. Moreover, the pairing dictates both the substrate choice and the modification type. The molecular details of generic E3-E2 interactions are well established.

Small, N-terminal tags activate Parkin E3 ubiquitin ligase activity by disrupting its autoinhibited conformation.

Parkin is an E3 ubiquitin ligase, mutations in which cause Autosomal Recessive Parkinson's Disease. Many studies aimed at understanding Parkin function, regulation and dysfunction are performed using N-terminal epitope tags. We report here that the use of small tags such as FLAG, cMyc and HA, influence the physical stability and activity of Parkin in and out of cells, perturbing the autoinhibited native state of Parkin, resulting in an active-for-autoubiquitination species.

Structural analysis of human FANCL, the E3 ligase in the Fanconi anemia pathway.

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein "core complex" containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not.

Mammal-restricted elements predispose human RET to folding impairment by HSCR mutations.

The maturation of human RET is adversely affected by a range of missense mutations found in patients with Hirschsprung's disease (HSCR), a complex multigenic disease. Here we show that two N-terminal cadherin-like domains, CLD1 and CLD2 (CLD(1-2)), from human RET adopt a clam-shell arrangement distinct from that of classical cadherins. CLD1 structural elements and disulfide composition are unique to mammals, indicating an unexpected structural diversity within higher and lower vertebrate RET CLD regions.

Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe.

We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome providing a tool for studying eukaryotic biology. Comprehensive gene dispensability comparisons with budding yeast--the only other eukaryote for which a comprehensive knockout library exists--revealed that 83% of single-copy orthologs in the two yeasts had conserved dispensability.

The structure of the catalytic subunit FANCL of the Fanconi anemia core complex.

The Fanconi anemia (FA) pathway is activated in response to DNA damage, leading to monoubiquitination of the substrates FANCI and FANCD2 by the FA core complex. Here we report the crystal structure of FANCL, the catalytic subunit of the FA core complex, at 3.2 A.

The RecB nuclease domain binds to RecA-DNA filaments: implications for filament loading.

The E. coli RecBCD enzyme facilitates the loading of RecA onto single-stranded DNA produced by the combined helicase/nuclease activity of RecBCD. The nuclease domain of RecB protein, RecB(nuc), has been previously shown to bind RecA.

Mechanistic basis of 5'-3' translocation in SF1B helicases.

Superfamily 1B (SF1B) helicases translocate in a 5'-3' direction and are required for a range of cellular activities across all domains of life. However, structural analyses to date have focused on how SF1A helicases achieve 3'-5' movement along nucleic acids. We present crystal structures of the complex between the SF1B helicase RecD2 from Deinococcus radiodurans and ssDNA in the presence and absence of an ATP analog.

ORC proteins: marking the start.

The DNA replication apparatus of archaea is more closely related to that of eukaryotes than eubacteria. Furthermore, recent work has shown that archaea, like eukaryotes, have multiple replication origins. Biochemical data are starting to reveal how archaeal origin binding proteins recognise and remodel origin DNA sequences.

Biochemical characterization of the minichromosome maintenance (MCM) protein of the crenarchaeote Aeropyrum pernix and its interactions with the origin recognition complex (ORC) proteins.

Replication in archaea is carried out by proteins that are homologues of eukaryotic counterparts. However, the archaeal systems tend to be much simpler with fewer different genes encoding the core functions than in eukaryotic counterparts. In many archaea, there is a single minichromosome maintenance (MCM) homologue, presumed to be the replicative helicase and between one and three origin recognition complex (ORC) homologues involved in binding to the replication origins.

DNA binding to RecD: role of the 1B domain in SF1B helicase activity.

The molecular mechanism of superfamily 1Balpha helicases remains unclear. We present here the crystal structure of the RecD2 helicase from Deinococcus radiodurans at 2.2-A resolution.

RecBCD: the supercar of DNA repair.

The DNA helicase RecBCD pauses when it reaches recombination hotspots known as Chi sites and then proceeds at a slower speed of translocation than before Chi recognition. Reporting in this issue, Spies et al. (2007) now show that this reduction in translocation velocity occurs when RecBCD changes which of its two motor subunits is in the lead.

Replication blocking lesions present a unique substrate for homologous recombination.

Homologous recombination (HR) plays a critical role in the restart of blocked replication forks, but how this is achieved remains poorly understood. We show that mutants in the single Rad51 paralog in Caenorhabditis elegans, rfs-1, permit discrimination between HR substrates generated at DNA double-strand breaks (DSBs), or following replication fork collapse from HR substrates assembled at replication fork barriers (RFBs). Unexpectedly, RFS-1 is dispensable for RAD-51 recruitment to meiotic and ionizing radiation (IR)-induced DSBs and following replication fork collapse, yet, is essential for RAD-51 recruitment to RFBs formed by DNA crosslinking agents and other replication blocking lesions.

Structure and mechanism of helicases and nucleic acid translocases.

Helicases and translocases are a ubiquitous, highly diverse group of proteins that perform an extraordinary variety of functions in cells. Consequently, this review sets out to define a nomenclature for these enzymes based on current knowledge of sequence, structure, and mechanism. Using previous definitions of helicase families as a basis, we delineate six superfamilies of enzymes, with examples of crystal structures where available, and discuss these structures in the context of biochemical data to outline our present understanding of helicase and translocase activity.

Redox-mediated substrate recognition by Sdp1 defines a new group of tyrosine phosphatases.

Reactive oxygen species trigger cellular responses by activation of stress-responsive mitogen-activated protein kinase (MAPK) signalling pathways. Reversal of MAPK activation requires the transcriptional induction of specialized cysteine-based phosphatases that mediate MAPK dephosphorylation. Paradoxically, oxidative stresses generally inactivate cysteine-based phosphatases by thiol modification and thus could lead to sustained or uncontrolled MAPK activation.

HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability.

Here, we show that the human homologue of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation.

Cellular functions of the BRCA tumour-suppressor proteins.

Inherited germline mutations in either BRCA1 or BRCA2 confer a significant lifetime risk of developing breast or ovarian cancer. Defining how these two genes function at the cellular level is essential for understanding their role in tumour suppression. Although BRCA1 and BRCA2 were independently cloned over 10 years ago, it is only in the last few years that significant progress has been made towards understanding their function in cells.

BRCA1 ubiquitylation of CtIP: Just the tIP of the iceberg?

Ubiquitylation is an important regulatory mechanism of many cellular processes. The breast and ovarian cancer-specific tumour suppressor BRCA1 is well acknowledged to be a RING/E3 ubiquitin ligase, however, identification of its physiological substrates has proved elusive. Recently published data have shown that the BRCA1-interacting protein CtIP is in fact ubiquitylated by BRCA1, and opens new avenues for the isolation of other substrate proteins.

Biochemical analysis of a DNA replication origin in the archaeon Aeropyrum pernix.

We have characterised the interaction of the Aeropyrum pernix origin recognition complex proteins (ORC1 and ORC2) with DNA using DNase I footprinting. Each protein binds upstream of its respective gene. However, ORC1 protein alone interacts more tightly with an additional region containing multiple origin recognition box (ORB) sites that we show to be a replication origin.

C. elegans FANCD2 responds to replication stress and functions in interstrand cross-link repair.

One of the least well understood DNA repair processes in cells is the repair of DNA interstrand cross-links (ICLs) which present a major obstacle to DNA replication and must be repaired or bypassed to allow fork progression. Fanconi anemia (FA) is an inherited genome instability syndrome characterized by hypersensitivity to ICL damage. Central to the FA repair pathway is FANCD2 that is mono-ubiquitylated in response to replication stress and ICL damage through the action of the FA core complex and its E3-ubiquitin ligase subunit, FANCL.

BRCA1-mediated ubiquitylation.

The BRCA1 tumour suppressor and its heterodimeric partner BARD1 play crucial roles in coordinating cellular responses to DNA damage. Evidence also implicates these proteins in transcriptional regulation, cell cycle progression and meiotic sex chromosome inactivation, but their mode of action remains elusive. The demonstration that the BRCA1/BARD1 heterodimer constitutes an E3-ubiquitin (Ub) ligase raises the possibility that ubiquitylation of specific targets may allow BRCA1/BARD1 to impact on diverse cellular processes.

CeBRC-2 stimulates D-loop formation by RAD-51 and promotes DNA single-strand annealing.

The BRCA2 tumour suppressor regulates the RAD-51 recombinase during double-strand break (DSB) repair by homologous recombination (HR) but how BRCA2 executes its functions is not well understood. We previously described a functional homologue of BRCA2 in Caenorhabditis elegans (CeBRC-2) that binds preferentially to single-stranded DNA via an OB-fold domain and associates directly with RAD-51 via a single BRC domain. Consistent with a direct role in HR, Cebrc-2 mutants are defective for repair of meiotic and radiation-induced DSBs due to an inability to regulate RAD-51.