Here, we describe the assembly of the nucleotide excision repair (NER) complex in normal and repair-deficient (xeroderma pigmentosum) human cells, employing a novel technique of local UV irradiation combined with fluorescent antibody labeling. The damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors in the preincision complex, including transcription repair factor TFIIH. XPA associates relatively late, is required for anchoring of ERCC1-XPF, and may be essential for activation of the endonuclease activity of XPG. These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight into the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of repair proteins at the site of the damage rather than a preassembled repairosome.

Abstract. Several nuclear activities and components are concentrated in discrete nuclear compartments. To understand the functional significance of nuclear compartmentalization, knowledge on the spatial distribution of transcriptionally active chromatin is essential. We have examined the distribution of sites of transcription by RNA polymerase II (RPII) by labeling nascent RNA with 5-bromouridine 5'-triphosphate, in vitro and in vivo. Nascent RPII transcripts were found in over 100 defined areas, scattered throughout the nucleoplasm. No preferential localization was observed in either the nuclear interior or the periphery. Each transcription site may represent the activity of a single gene or, considering the number of active pre-mRNA genes in a cell, of a cluster of active genes. The relation between the distribution of nascent RPII transcripts and that of the essential splicing factor SC-35 was investigated in double labeling experiments. Antibodies against SC-35 recognize a number of welldefined, intensely labeled nuclear domains, in addition to labeling of more diffuse areas between these domains (Spector, D. L., X. -D. Fu, and T. Maniatis.1991. EMBO (Eur. Mol. Biol. Organ.)J. 10:3467-3481). We observe no correlation between intensely labeled SC-35 domains and sites of pre-mRNA synthesis. However, many sites of RPII synthesis colocalize with weakly stained areas. This implies that cotranscriptional splicing takes place in these weakly stained areas. These areas may also be sites where splicing is completed posttranscriptionally. Intensely labeled SC-35 domains may function as sites for assembly, storage, or regeneration of splicing components, or as compartments for degradation of introns.T HE cell nucleus comprises all factors required for faithful replication of the genome and regulated synthesis, processing and transport of RNA. In recent years much information on nuclear organization has become available. It is clear now that the nucleus is highly organized (reviewed by Jackson, 1991; van Driel et al., 1991). The most conspicuous subnuclear domain is the nucleolus in which ribosomal genes from different chromosomes are clustered and ribosome assembly takes place (reviewed by Scheer and Benavente, 1990; HernandezVerdun, 1991). Other examples of a domainlike organization in the nucleus are: replication clusters during S-phase (reviewed by Berezney, 1991), clustered splicing components (Spector, 1990;Fu and Maniatis, 1990; CarmoFonseca et al., 1992), hnRNP proteins (Pifiol-Roma et al., 1989; Ghetti et al., 1992), and tracks and foci of specific RNAs (Lawrence and Singer, 1991; Huang and Spector, 1991). In addition, a number of structures have been visualized of which the function is still unknown (Ascoli and Maul, 1991;Saunders et al., 1991; Ra~ka et al., 1991; al., 1992). The structural basis of the occurrence of nuclear activities in domains and the functional significance of this organizing principle for the regulation of gene expression and DNA replication are not understood.Essential for understandi...

Genome function in higher eukaryotes involves major changes in the spatial organization of the chromatin fiber. Nevertheless, our understanding of chromatin folding is remarkably limited. Polymer models have been used to describe chromatin folding. However, none of the proposed models gives a satisfactory explanation of experimental data. In particularly, they ignore that each chromosome occupies a confined space, i.e., the chromosome territory. Here, we present a polymer model that is able to describe key properties of chromatin over length scales ranging from 0.5 to 75 Mb. This random loop (RL) model assumes a self-avoiding random walk folding of the polymer backbone and defines a probability P for 2 monomers to interact, creating loops of a broad size range. Model predictions are compared with systematic measurements of chromatin folding of the q-arms of chromosomes 1 and 11. The RL model can explain our observed data and suggests that on the tens-of-megabases length scale P is small, i.e., 10 -30 loops per 100 Mb. This is sufficient to enforce folding inside the confined space of a chromosome territory. On the 0.5-to 3-Mb length scale chromatin compaction differs in different subchromosomal domains. This aspect of chromatin structure is incorporated in the RL model by introducing heterogeneity along the fiber contour length due to different local looping probabilities. The RL model creates a quantitative and predictive framework for the identification of nuclear components that are responsible for chromatin-chromatin interactions and determine the 3-dimensional organization of the chromatin fiber.genome organization ͉ polymer model ͉ chromatin folding T he chromatin fiber inside the interphase nucleus of higher eukaryotes is folded and compacted on several length scales. On the smallest scale the basic filament is formed by wrapping double-stranded DNA around a histone protein octamer, forming a nucleosomal unit every Ϸ200 bp. This beads-on-a-string type filament in turn condenses to a fiber of 30-nm diameter, which detailed organization is still under debate (1-3). At bigger length scales the spatial organization of chromatin in the interphase nucleus is even more unclear. Imaging techniques do not allow one to directly follow the folding path of the chromatin fiber in the interphase nucleus. Therefore, indirect approaches have been used to obtain information about chromatin folding. One way, pursued in this study, is fluorescence in situ hybridization (FISH) to measure the relationship between the physical distance between genomic sequence elements (in m) and their genomic distance (in megabases). There have been several attempts to explain the folding of chromatin in the interphase nucleus using polymer models. The strength of polymer models is their ability to make predictions on the structure of chromatin by pointing out the driving forces for observed folding motifs. These predictions can then be tested experimentally. However, a polymer model that is able to explain chromatin folding spanning differen...

Heterochromatin protein 1 (HP1) family members are chromatin-associated proteins involved in transcription, replication, and chromatin organization. We show that HP1 isoforms HP1-α, HP1-β, and HP1-γ are recruited to ultraviolet (UV)-induced DNA damage and double-strand breaks (DSBs) in human cells. This response to DNA damage requires the chromo shadow domain of HP1 and is independent of H3K9 trimethylation and proteins that detect UV damage and DSBs. Loss of HP1 results in high sensitivity to UV light and ionizing radiation in the nematode Caenorhabditis elegans, indicating that HP1 proteins are essential components of DNA damage response (DDR) systems. Analysis of single and double HP1 mutants in nematodes suggests that HP1 homologues have both unique and overlapping functions in the DDR. Our results show that HP1 proteins are important for DNA repair and may function to reorganize chromatin in response to damage.