NBS1 recruits RAD18 via a RAD6-like domain and regulates Pol η-dependent translesion DNA synthesis

Translesion DNA synthesis, a process orchestrated by monoubiquitinated PCNA, is critical for DNA damage tolerance. While the ubiquitin-conjugating enzyme RAD6 and ubiquitin ligase RAD18 are known to monoubiquitinate PCNA, how they are regulated by DNA damage is not fully understood. We show that NBS1 (mutated in Nijmegen breakage syndrome) binds to RAD18 after UV irradiation and mediates the recruitment of RAD18 to sites of DNA damage. Disruption of NBS1 abolished RAD18-dependent PCNA ubiquitination and Polη focus formation, leading to elevated UV sensitivity and mutation. Unexpectedly, the RAD18-interacting domain of NBS1, which was mapped to its C terminus, shares structural and functional similarity with the RAD18-interacting domain of RAD6. These domains of NBS1 and RAD6 allow the two proteins to interact with RAD18 homodimers simultaneously and are crucial for Polη-dependent UV tolerance. Thus, in addition to chromosomal break repair, NBS1 plays a key role in translesion DNA synthesis.     

A molecular mechanism for DNA damage recognition by the xeroderma pigmentosum group C protein complex

The XPC-HR23B complex is involved in DNA damage recognition and the initiation of global genomic nucleotide excision repair (GG-NER). Our previous studies demonstrate that XPC-HR23B recognizes and binds DNA containing a helix distortion, regardless of the presence or absence of damaged bases. Here, we describe an extended analysis of the DNA binding specificity of XPC-HR23B using various defined DNA substrates. Although XPC-HR23B showed significantly higher affinity for single-stranded DNA than double-stranded DNA, specific secondary structures of DNA, involving a single- and double-strand junction, were strongly preferred by the complex. This indicates that the presence of bases, which cannot form normal Watson-Crick base pairs in double-stranded DNA, is a critical factor in determining the specificity of XPC-HR23B binding. A DNase I footprint analysis, using a looped DNA substrate, revealed that a single XPC-HR23B complex protected a distorted site in an asymmetrical manner, consistent with the preferred secondary structure. The specific binding of XPC-HR23B is undoubtedly an important molecular process, based on which NER machinery detects a wide variety of lesions that vary in terms of chemical structure during DNA repair.     

Ultrastructural Changes in Cortical Cells of Apple (Malus pumila Mill.) Associated with Cold Hardiness

Seasonal ultrastructural changes in cortical cells of apple(Malus pumila Mill.) twigs were studied with special referenceto seasonal variations in cold hardiness. The ultrastructuralcharacteristics could be divided into two major sets: one setthat developed during cold acclimation from September to Januaryand one that developed during deacclimation from February toMay. During cold acclimation, the most striking changes weremicrovacuolation and augmentation of the volume of the cytoplasm.At this stage, the cells became temporarily rich in the organellesthat are involved in protein synthesis, such as vesicular endoplasmicreticulum, polysomes, dictyosomes and vesicles. Plastids thatcontained starch granules, protein-lipid bodies and mitochondriawere also abundant. Each nucleus contained relatively loweramounts of heterochromatin and was located in the central portionof the cell. From mid-November until March, plastids aggregatedaround the nucleus, and the formation of "plastid initials"from the mature plastids, as a results of constriction and subsequentpinching off, was frequently observed. In January, when maximumcold-hardiness was achieved, the starch granules in plastidsdisappeared. The second set of ultrastructural changes, whichincluded fusion of vacuoles, was initiated in late February.During deacclimation, the differentiation of vacuoles proceededin the cells, starch granules reappeared in the plastids andorganelles involved in protein synthesis became abundant. Furthermore,vesicular endoplasmic reticulum observed during the autumn andwinter was replaced by smooth endoplasmic reticulum. In mid-May,when cold hardiness decreased to a low level, most of the cellspace was occupied by a large vacuole. The results suggest that seasonal cytological changes are involvedin the changes in the physical and functional properties ofcold-adapted cells. The seasonal replication of vacuoles andcytoplasm may be related to resistance or susceptibility tothe stress-producing effects of the dehydration that occursduring freezing, and the replication of organelles may be associatedwith the metabolism required for cold acclimation or deacclimation. (Received December 3, 1992; Accepted December 28, 1993)     

Occurrence of unidentified bodies that resemble “plastid initials” in poplar cells after breaking of dormancy in midwinter

Summary Electron-microscopic studies of plastids in cortical cells of poplar (Populus euramericana cv. gelrica) were carried out to examine whether any structural changes were initiated after breaking of dormancy in midwinter under non-growing conditions. After the breaking of dormancy, ultrastructural changes became evident and the profiles of plastids became heterogeneous. Organelles resembling the plastid initials proposed by Mühlenthaler and Frey-Wyssling in 1965 were frequently observed concomitant with changes in the plastid envelope. The formation of plastid initials appeared to be initiated by the formation of septa in pre-existing plastids. After this stage, narrow connections appeared between the initials and the parent plastids. Approximately 50 days after the breaking of dormancy in late March, further heterogeneity in the profiles of plastids was observed. At this stage, young plastids (plastids without starch granules) were frequently observed and the formation of plastid initials was hardly ever observed. These observations suggest that the plastid initials may be present for only a limited period in the cortical cells of the poplar and may be the precursors of the proplastids. Similar ultrastructural profiles were found in cortical cells of mulberry and in leaf buds of apple trees, suggesting that such changes in the ultrastructure of plastids are a general feature of perennials.     

The proliferation of amyloplasts in meristematic cells of developing stolons of potato and apple callus: progenitors of proplastids

To monitor the events in the proliferation of amyloplasts, the ultrastructure of relevant structures in the cytosol has to be studied. For this investigation, photographs of cellular ultrastructures in developing potato stolons and apple callus were taken and examined. The images indicated that the contribution to proliferation of the division of mature amyloplasts was extremely low and that the major pathway involved the generation of the proplastids from "mother" amyloplasts. The generation of proplastids was followed either by division into small bodies of 1microm or less in diameter or by growth to slender proplastids of 5microm in length. The elongated proplastids multiplied by splitting at random sites, with subsequent enlargement to mature sizes. The latter process contributed to the massive accumulation of amyloplasts in cells but has not previously been adequately emphasized. With respect to the putative "mother" amyloplasts, numerous divergent amyloplasts were observed with a considerably different ultrastructure compared to the normal types, and with a characteristically extended and constricted stroma. Various lines of evidence indicated that the divergent amyloplasts were the "mother" amyloplasts of the proplastids. No other plastidic organelles with features that suggest the generation of proplastids were detected in the cytosol.     

An EF-hands protein, centrin-1, is an EGTA-sensitive SUMO-interacting protein in mouse testis

A multifunctional calcium‐binding protein, centrin‐1, is specifically expressed in male germ cells, certain neurons and ciliated cells. We identified centrin‐1 as a protein interacting with SUMO‐2/3 using yeast two‐hybrid screening of a mouse testicular cDNA library. In bead halo assays, the interaction between centrin‐1 and SUMO‐2/3 was reduced in the presence of EGTA and facilitated by the addition of CaCl_2. immunostaining of seminiferous tubules in 35‐day‐old mouse testes revealed that cells in the layer containing spermatogonia showed colocalization of SUMO‐2/3 with centrin‐1 in cytoplasmic spots. Identification of centrin‐1 as the EGTA‐sensitive SUMO‐2/3‐interacting protein indicates the possible role of calcium in modulating the centrin‐1–SUMO‐2/3 interaction and suggests the importance of this interaction in mouse testis. Copyright © 2010 John Wiley & Sons, Ltd.     

Molecular analysis of a novel hereditary C3 deficiency with systemic lupus erythematosus

A case of inherited homozygous complement C3 deficiency (C3D) in a patient with systemic lupus erythematosus (SLE) and the molecular basis for this deficiency are reported. A 22-year-old Japanese male was diagnosed as having SLE and his medical history revealed recurrent tonsillitis and pneumonia. He was diagnosed as having C3D because of undetectable serum C3 level. His parents were consanguineous. Sequence analysis of C3D cDNA revealed a homozygous deletion of exon 39 (84bp). A single base substitution (AG to GG) in the 3'-splice acceptor site of intron 38 was identified by sequencing the genomic DNA. Expression of C3Delta(ex39) cDNA, the C3cDNA lacking exon 39, in COS-7 cells revealed that C3Delta(ex39) was retained in endoplasmic reticulum-Golgi intermediate compartment because of defective secretion. These data indicate that a novel AG-->GG 3'-splice acceptor site mutation in intron 38 caused aberrant splicing of exon 39, resulting in defective secretion of C3.