Nuclear Rad51 focus formation is required for homologydirected repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions.

Background:The regulation of multi-functional DYNLL1 is poorly understood. Results: ASCIZ activates Dynll1 gene expression and is inhibited by DYNLL1 binding to its transcription activation domain. Conclusion: ASCIZ plays a key role in the auto-regulation of DYNLL1 levels. Significance: This is the first case where a gene product directly inhibits its main transcriptional activator while bound at its own promoter.

Mutations in the LMNA gene, which encodes all A-type lamins, including lamin A and lamin C, cause a variety of tissue-specific degenerative diseases termed laminopathies. Little is known about the pathogenesis of these disorders. Previous studies have indicated that A-type lamins interact with the retinoblastoma protein (pRB). Here we probe the functional consequences of this association and further examine links between nuclear structure and cell cycle control. Since pRB is required for cell cycle arrest by p16 ink4a , we tested the responsiveness of multiple lamin A/C-depleted cell lines to overexpression of this CDK inhibitor and tumor suppressor. We find that the loss of A-type lamin expression results in marked destabilization of pRB. This reduction in pRB renders cells resistant to p16 ink4a -mediated G 1 arrest. Reintroduction of lamin A, lamin C, or pRB restores p16 ink4a -responsiveness to Lmna ؊/؊ cells. An array of lamin A mutants, representing a variety of pathologies as well as lamin A processing mutants, was introduced into Lmna ؊/؊ cells. Of these, a mutant associated with mandibuloacral dysplasia (MAD R527H), as well as two lamin A processing mutants, but not other disease-associated mutants, failed to restore p16 ink4a responsiveness. Although our findings do not rule out links between altered pRB function and laminopathies, they fail to support such an assertion. These findings do link lamin A/C to the functional activation of a critical tumor suppressor pathway and further the possibility that somatic mutations in LMNA contribute to tumor progression.A-type lamins are intermediate filament proteins that have been linked to the organization and maintenance of nuclear structure. In most differentiated tissues, A-type lamins (predominantly lamins A and C), along with lamin B, comprise the meshwork underlying the inner nuclear membrane known as the nuclear lamina (63). Unlike lamin C, lamin A contains a carboxy-terminal CaaX motif and must undergo a series of posttranslational modifications to form the mature lamin A (77). In particular, the cysteine of the CaaX motif in lamin A is isoprenylated, followed by cleavage of the aaX and carboxymethylation of the C-terminal cysteine (72). Lastly, a second cleavage event by Zmpste24 removes the last 15 amino acids to yield the mature lamin A (3, 13, 74).Mutations within the LMNA gene cause a variety of human disorders collectively known as laminopathies. These include progeria syndromes and dystrophies associated with skeletal muscle and adipose tissue (5,10,15,19,45,58). Mice lacking A-type lamins or deficient in processing lamin A develop skeletal and cardiac muscular dystrophies, thus confirming the importance of A-type lamins for muscle differentiation and/or maintenance (52, 65). The mechanism by which mutations in A-type lamins generate tissue-specific disorders is unknown. However, one model posits that lamin A/C regulates gene expression in differentiated tissue by coordinating the activity of key transcription factors.A-type lamins localize to p...

Zn2+-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn2+-finger protein (ASCIZ; also known as ATMIN and ZNF822) was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion or knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis.

SummaryForkhead-associated (FHA) domains are present in 4200 diverse proteins in all phyla from bacteria to mammals and seem to be particularly prevalent in proteins with cell cycle control functions. Recent work from several laboratories has considerably improved our understanding of the structure and function of these domains that were virtually unknown a few years ago, and the first disease associations of FHA domains have now emerged. FHA domains form 11-stranded beta-sandwiches that contain some 100-180 amino acid residues with a high degree of sequence diversity. FHA domains act as phosphorylation-dependent protein-protein interaction modules that preferentially bind to phospho-threonine residues in their targets. Interestingly, point mutations in the human CHK2 gene that lead to single-residue amino acid substitutions in the FHA domain of this cell cycle checkpoint kinase have been found to cause a subset of cases of the Li-Fraumeni multi-cancer syndrome.

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are present in variable number in most human cell types and have been linked to various cellular functions, including roles as depots for DNA repair proteins. Here, we show that treatment of human cells with DNA methylating agents leads to redistribution of PML from NBs to a diffuse nuclear localization. Biochemically, this correlates with a specific reduction of PML levels in the nuclear matrix fraction without affecting total PML levels. Similar results were obtained for the other major PML NB component, the Sp100 protein, indicating that DNA methylating agents lead to a general disassembly of PML NBs. Similar to the dispersal of PML NBs in response to some viral infections, PML redistribution after DNA damage was inhibited by the proteasome inhibitor MG132. We propose that the regulated dispersal of PML NBs may facilitate the enhanced release of DNA repair proteins from NB depots in order to respond adequately to extensive DNA damage.

Parathyroid hormone-related protein (PTHrP) has a diverse range of proposed biological activities participating in both extracellular and intracellular signaling. In order to identify candidate protein e¡ectors, yeast two-hybrid screens were conducted using mature human PTHrP (residues 1^141) and the COOH-terminus (residues 107^141). Both PTHrP baits interacted with a L L-arrestin 1B fragment, an important component of G-protein-coupled receptor desensitization and MAPK signaling. Co-immunoprecipitation, in vitro binding assays and colocalization experiments con¢rmed this interaction in human cells and this required residues 122^141 of PTHrP. These ¢ndings suggest that L L-arrestin 1 acts as an e¡ector for a novel function of PTHrP in cytoplasm. ß

No abstract