To study initiation of DNA replication in mammalian chromosomes, we have established a methotrexateresistant Chinese hamster ovary cell line (CHOC 400) that contains -1,000 copies of the early replicating dihydrofolate reductase (DHFR) domain. We have previously shown that DNA replication in the prevalent 243-kilobase (kb) amplicon type in this cell line initiates somewhere within a 28-kb region located downstream from the DHFR gene. In an attempt to localize the origin of replication with more precision, we blocked the progress of replication forks emanating from origins at the beginning of the S phase by the introduction of trioxsalen cross-links at 1-to 5-kb intervals in the parental double-stranded DNA. The small DNA fragments synthesized under these conditions (which should be centered around replication origins) were then used as hybridization probes on digests of cosmids and plasmids from the DHFR domain. These studies suggested that in cells synchronized by this regimen, DNA replication initiates at two separate sites within the previously defined 28-kb replication initiation locus, in general agreement with results described in the accompanying paper (T.-H. Leu and J. L. Hamlin, Mol. Cell. Biol. 9:523-531, 1989). One of these sites contains a repeated DNA sequence element that is found at or near many other initiation sites in the genome, since it was also highly enriched in the early replicating DNA isolated from cross-linked CHO cells that contain only two copies of the DHFR domain.

In higher eukaryotic cells, DNA is tandemly arranged into 10 4 replicons that are replicated once per cell cycle during the S phase. To achieve this, DNA is organized into loops attached to the nuclear matrix. Each loop represents one individual replicon with the origin of replication localized within the loop and the ends of the replicon attached to the nuclear matrix at the bases of the loop. During late G 1 phase, the replication origins are associated with the nuclear matrix and dissociated after initiation of replication in S phase. Clusters of several replicons are operated together by replication factories, assembled at the nuclear matrix. During replication, DNA of each replicon is spooled through these factories, and after completion of DNA synthesis of any cluster of replicons, the respective replication factories are dismantled and assembled at the next cluster to be replicated. Upon completion of replication of any replicon cluster, the resulting entangled loops of the newly synthesized DNA are resolved by topoisomerases present in the nuclear matrix at the sites of attachment of the loops. Thus, the nuclear matrix plays a dual role in the process of DNA replication: on one hand, it represents structural support for the replication machinery and on the other, provides key protein factors for initiation, elongation, and termination of the replication of eukaryotic DNA. J. Cell. Biochem. 96: 951-961, 2005. ß 2005Wiley-Liss,Inc.

Chromatin modifications/remodeling are important mechanisms by which cells regulate various functions through providing accessibility to chromatin DNA. Recent studies implicated INO80, a conserved chromatinremodeling complex, in the process of DNA repair. However, the precise underlying mechanism by which this complex mediates repair in mammalian cells remains enigmatic. Here, we studied the effect of silencing of the Ino80 subunit of the complex on double-strand break repair in mammalian cells. Comet assay and homologous recombination repair reporter system analyses indicated that Ino80 is required for efficient double-strand break repair. Ino80 association with chromatin surrounding double-strand breaks suggested the direct involvement of INO80 in the repair process. Ino80 depletion impaired focal recruitment of 53BP1 but did not impede Rad51 focus formation, suggesting that Ino80 is required for the early steps of repair. Further analysis by using bromodeoxyuridine (BrdU)-labeled single-stranded DNA and replication protein A (RPA) immunofluorescent staining showed that INO80 mediates 5-3 resection of double-strand break ends.Efficient repair of DNA double-strand breaks (DSBs) is required to prevent genomic instability and subsequent oncogenic transformation. Repair of DSBs is carried out by nonhomologous end joining (NHEJ) and homologous recombination (HR) (10,37,46). These processes are confronted with DNA being packaged into nucleosomes, which presents a natural barrier to the access of the repair machinery. Thus, to carry out repair, chromatin surrounding DNA breaks needs to be modified and remodeled. While histone modifications have been recognized as important to provide accessibility to DNA lesions and binding interfaces for the recruitment and retention of repair factors (15), studies in yeast have suggested that chromatin remodeling may also play an important role in repair. Chromatin-remodeling complexes use the energy of ATP hydrolysis to alter histone-DNA interactions and to reposition nucleosomes along DNA. The yeast INO80 chromatin-remodeling complex has been the most studied in this respect. Yeast strains deleted for subunits of the INO80 complex are hypersensitive to DSB-inducing agents (35). There is evidence suggesting that INO80 directly participates in both HR and NHEJ repair pathways (20,40,42,43) and facilitates HR-mediated recovery of stalled replication forks (24,36). In addition, a study by Wu et al. (45) showed that depletion of the Polycomb group protein Yin Yang1 (YY1) and/or Ino80 impaired UVmediated DNA damage response and reduced HR rates in mammalian cells, suggesting that YYI mediates HDR through its interaction with INO80 complex, although the precise molecular events involving YY1-INO80 that govern HR repair have not been revealed. However, there are ambiguous results about its role in HR and chromatin processing of DSB ends. Thus, it has been shown that in Saccharomyces cerevisiae, the INO80 complex participated in nucleosome eviction in the vicinity of a DSB at the MAT loc...