Repair of DNA in replicated and unreplicated portions of the human genome.

Portions of the human genome that have replicated after ultraviolet light irradiation and those that remain unreplicated have both been examined for the distribution of pyrimidine dimers and the extent of repair replication following their removal. The data indicate that the number of unrepaired dimers and the extent of repair replication seen after their excision are equal in the replicated and unreplicated DNA. Furthermore, the daughter strand of replicated DNA is larger than the average interdimer distance found in the parental strand. Hence, DNA replication in normal human fibroblasts is clearly capable of getting past pyrimidine dimers, and a preferential repair of such lesions in DNA that is about to be or has been replicated does not operate to any visible extent in these cells.     

Low-level DNA exchanges in normal human and xeroderma pigmentosum cells after UV irradiation

We have used a T4 endonuclease V assay method for UV-induced pryrimidine dimers in cellular DNA in vivo to obtain evidence for recombinational DNA exchanges after UV irradiation of normal human and Xeroderma pigmentosum (XP) cells. Our data indicate that the endonuclease-sensitive sites in excision-defective XP cells are removed very slowly from the irradiated parental strands and appear concomitantly in daughter strands newly synthesized during post-UV incubation. In the defective XP cells, the extent of appearance of sensitive sites in daughter strands synthesized during a period of 24 h after 10 J/m² appears to be small, probably less than 15% of the initial number of sensitive sites detected in cellular parental strands. Demonstration of such exchanges between normal-density parental and 5-bromodeoxyuridine-labeled daughter strands by alkaline CsCl isopycnic centrifugation was unsuccessful. Further, the extent is much lower in normal human cell because of their efficiet excision repair of the dimers before and after exchanges than in the defective XP cells.     

Evidence that dimers remaining in preinduced Escherichia coli B/r Hcr+ become insensitive after DNA replication to the extract from Micrococcus luteus.

In Escherichia coli B/r Her+ irradiated with two separate fluences, dimer excision is prematurely interrupted. The present study was designed to follow tha fate of dimers remaining unexcised. The results imply that these dimers (or distortions containing dimers) are transformed on replication from the state of sensitivity to the state of insensitivity to endonuclease from Micrococcus luteus. This conclusion is based on the following findings: (a) dimers were radiochromatographically detectable in DNA replicated after UV, which indicated that they were tolerated on replication. (b) Similar amounts of dimers were detected radiochromatographically both in DNA remaining unreplicated and DNA twice replicated after UV, This along with the low transfer of parental label into daughter DNA, indicated that dimers remained in situ in parental chains. (c) Immediately after UV, all parental DNA contained numerous sites sensitive to the extract from M. luteus. 2 h after UV, a portion of parental DNA still contained a number of endonuclease-sensitive (Es) sites, while another portion of parental DNA and all daughter DNA were free of Es sites. (d) The occurrence of parental DNA free of Es sites was not temporally correlated with dimer excision, but with the first round of DNA replication. (e) The amount of DNA free of Es sites corresponded to the amount of replicated DNA. (f) Separation of replicated and unreplicated DNA, and detection of Es sites in both portions separately showed that the replicated DNA was almost free of Es sites, whereas unreplicated DNA contained a number of such sites.     

Differential repair and replication of damaged DNA in ribosomal RNA genes in different CHO cell lines

We studied the repair of psoralen adducts in the pol I-transcribed ribosomal RNA (rRNA) genes of excision repair competent Chinese hamster ovary (CHO) cell lines, their UV sensitive mutant derivatives, and their UV resistant transformants, which express a human excision repair gene. In the parental cell line CHO-AA8, both monoadducts and interstrand crosslinks are removed efficiently from the rRNA genes, whereas neither adduct is removed in the UV sensitive derivative UV5; removal of both adducts is restored in the UV resistant transformant CHO-5T4 carrying the human excision repair gene ERCC-2. In contrast, removal of psoralen adducts from the rRNA genes is not detected in another parental CHO cell line CHO-9, neither in its UV sensitive derivative 43-3B, nor in its UV resistant transformant 83-G5 carrying the human excision repair gene ERCC-1. In contrast to such intergenomic heterogeneity of repair, persistence of psoralen monoadducts during replication of the rRNA genes occurs equally well in all CHO cell lines tested. From these data, we conclude that: 1) the repair efficiency of DNA damage in the rRNA genes varies between established parental CHO cell lines; 2) the repair pathways of intrastrand adducts and interstrand crosslinks in mammalian cells share, at least, one gene product, i.e., the excision repair gene ERCC-2; 3) replicational bypass of psoralen monoadducts at the CHO rRNA locus occurs similarly on both DNA strands.     

Replication in the amplified dihydrofolate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element.

To study initiation of DNA replication in mammalian chromosomes, we have established a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains approximately 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.     

Inhibition and recovery of DNA synthesis in human cells after exposure to ultraviolet light

The inhibition of DNA synthesis in normal human cells by UV is a complex function of fluence because it has several causes. At low fluences, inhibition of replicon initiation is most important. This is made clear by the fact that it occurs to a lesser degree in cells from patients with ataxia telangiectasia (AT). Assuming that only leading strand synthesis is blocked by UV-induced lesions, single lesions between replicons in parental strands for leading strand synthesis inhibit DNA synthesis by acting as temporary blocks until they are replicated by extension of the lagging strand of the adjacent replicon. A more severe inhibition occurs when two lesions are induced between adjacent growing replicons, because one in four possible configurations may result in a long-lived unreplicated region (LLUR). In the absence of excision repair, these may eventually be replicated by activation of an otherwise unused origin within the LLUR. The frequency of LLURs increases steeply with fluence. Activation of normally unused origins to replicate LLURs may facilitate recovery from inhibition of DNA synthesis, but repair of lesions is probably more important. In excision-repair-defective cells, an LLUR without an origin to initiate its replication may be a lethal lesion.     

The early and late modes of DNA replication in ultraviolet irradiated Syrian hamster embryo cells.

The nature of DNA replication in UV irradiated Syrian hamster embryo cells (HEC) was investigated by measuring the size distribution of nascent daughter strand DNA. During the early mode nascent strands are made in smaller pieces than in nonirradiated cells. The late mode begins when nascent strands recover to normal size. This was observed in HEC 5 h post-UV. When the late mode is operational, nascent strands elongate to parental size in greater than 2 h, whereas less than 3 h are required during early mode function. Evidence from split dose experiments demonstrates that the recovery of the size of nascent strands is not due to enhanced gap filling. Furthermore, pyrimidine dimers are probably recognized differently by the replication complex during early and late mode DNA synthesis. The late mode of replication could account for the ability of HEC to survive UV irradiation even though they are inefficient in both excision and postreplication repair.     

Preirradiation of host cells does not alter blockage of simian virus 40 replication forks by pyrimidine dimers

Do damage-inducible responses in mammalian cells alter the interaction of lesions with replication forks? We have previously demonstrated that preirradiation of the host cell mitigates UV inhibition of SV40 DNA replication; this mitigation can be detected within the first 30 min after the test irradiation. Here we test the hypotheses that this mitigation involves either (1) rapid dimer removal, (2) rapid synthesis of daughter strands past lesions (trans-dimer synthesis), or (3) continued progression of the replication fork beyond a dimer. Cells preirradiated with UV were infected with undamaged SV40, and the effects of UV upon viral DNA synthesis were measured within the first hour after a subsequent test irradiation. In preirradiated cells, as well as in non-preirradiated cells, pyrimidine dimers block elongation of daughter strands; daughter strands grow only to a size equal to the interdimer distance along the parental strands. There is, within this first hour after UV, no evidence for trans-dimer synthesis, nor for more rapid dimer removal either in the bulk of the parental DNA or in molecules in the replication pool. Progression of the replication forks was analyzed by electron microscopy of replicating SV40 molecules. Dimers block replication-fork progression in preirradiated cells to the same extent as in non-preirradiated cells. These experiments argue strongly against the hypotheses that preirradiation of host cells results in either the rapid removal of dimers, trans-dimer synthesis, or continued replication-fork progression beyond dimers.     

Intermediate in adenovirus type 2 replication.

Replicating chromosomes, called intermediate DNA, have been extracted from the adenovirus replication complex. Compared to mature molecules, intermediate DNA had a greater buoyant density in CsCl gradients and ethidium bromide-cesium chloride gradients. Digestion of intermediate DNA with S1 endonuclease, but not with RNase, abolished the difference in densities. These properties suggest that replicating molecules contain extensive regions of parental single strands. Although intermediate DNA sedimented faster than marker viral DNA in neutral sucrose gradients, single strands longer than unit length could not be detected after alkaline denaturation. Integral size classes of nascent chains in intermediate DNA suggest a relationship between units of replication and the nucleoprotein structure of the virus chromosome. Adenovirus DNA was replicated at a rate of 0.7 x 10-6 daltons/min. Although newly synthesized molecules had the same sedimentation coefficient and buoyant density as mature chromosomes, they still contained single-strand interruptions. Complete joining of daughter strands required an additional 15 to 20 min.     

Mapping of replication initiation sites in mammalian genomes by two-dimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix.

Two complementary two-dimensional gel electrophoretic techniques have recently been developed that allow initiation sites to be mapped with relative precision in eukaryotic genomes at least as complex as those of yeast and Drosophila melanogaster. We reported the first application of these mapping methods to a mammalian genome in a study on the amplified dihydrofolate reductase (DHFR) domain of the methotrexate-resistant CHO cell line CHOC 400 (J.P. Vaughn, P.A. Dijkwel, and J.L. Hamlin, Cell 61:1075-1087, 1990). Our results suggested that in this 240-kb domain, initiation of nascent DNA strands occurs at many sites within a 30- to 35-kb zone mapping immediately downstream from the DHFR gene. In the course of these studies, it was necessary to develop methods to stabilize replication intermediates against branch migration and shear. This report describes these stabilization methods in detail and presents a new enrichment protocol that extends the neutral/neutral two-dimensional gel mapping method to single-copy loci in mammalian cells. Preliminary analysis of replication intermediates purified from CHO cells by this method suggests that DNA synthesis may initiate at many sites within a broad zone in the single-copy DHFR locus as well.     

Transfer of nucleosomes from parental to replicated chromatin.

Simian virus 40 (SV40) minichromosomes were used as the substrate for in vitro replication. Protein-free SV40 DNA or plasmids, carrying the SV40 origin of replication, served as controls. Replicated minichromosomal DNA possessed constrained negative superhelicity indicative of the presence of nucleosomes. The topological state of replicated minichromosomal DNA was precisely determined by two-dimensional gel electrophoresis. We show that most or all nucleosomes, present on the replicated minichromosomal DNA, were derived from the parental minichromosome substrate. The mode and the rate of nucleosome transfer from parental to minichromosomal daughter DNA were not influenced by high concentrations of competing replicating and nonreplicating protein-free DNA, indicating that nucleosomes remain associated with DNA during the replication process. The data also show that parental nucleosomes were segregated to the replicated daughter DNA strands in a dispersive manner.     

Characterization of gene-specific DNA repair by primary cultures of rat hepatocytes

At present, almost all the information on gene-specific DNA repair in mammals comes from studies with transformed cell lines and proliferating primary cells obtained from rodents and humans. In the present study, we measured the repair of specific DNA regions in primary cultures of nondividing rat hepatocytes (parenchymal cells). DNA damage was induced by irradiating the primary cultures of hepatocytes with ultraviolet (UV) light, and the presence of cyclobutane pyrimidine dimers (CPDs) was measured by using T4 endonuclease V in the following: a 21-kb BamHI fragment containing the albumin gene, a 14-kb BamHI fragment containing the H-ras gene, and the genome overall. The frequency of CPDs in the two BamHI fragments and the genome overall were similar and ranged from 0.5 to 1.3 CPDs per 10 kb for UV doses of 5–30 J/m². However, the removal of CPDs from the DNA fragment containing the albumin gene was significantly higher than from that of the genome overall and the DNA fragment containing the H-ras gene. Within 24 hr, approximately 67% of the CPDs was removed from the DNA fragment containing the albumin gene versus less than 40% for the genome overall and the DNA fragment containing the H-ras gene. The lower repair observed for the 14-kb fragment containing the H-ras gene is probably indicative of repair of the nontranscribed region of this fragment because the H-ras gene makes up only 2.4 kb of the 14-kb fragment. Primary cultures of hepatocytes removed CPDs from the transcribed strand of albumin fragment more efficiently than from the nontranscribed strand; however, no differences were observed in the repair of the two strands of the fragment containing the H-ras gene. These results demonstrate that primary cultures of nondividing rat hepatocytes show differential repair of UV-induced DNA damage that is comparable to what has been reported for transformed, proliferating mammalian cell lines. J. Cell. Physiol. 176:314–322, 1998. © 1998 Wiley-Liss, Inc.     

The replication timing of the amplified dihydrofolate reductase genes in the Chinese hamster ovary cell line CHOC 400

We have examined the timing of replication of the amplified dihydrofolate reductase genes in the methotrexate-resistant Chinese hamster ovary cell line CHOC 400 using two synchronization procedures. DNA replicated in the presence of 5-bromodeoxyuridine was collected from cells of various times during the DNA synthesis phase and the extent of replication for defined sequences was determined by Southern blotting analysis of CsCl density gradient fractions. We report that under these conditions the DHFR gene replicates throughout the course of S phase in a mode similar to the bulk of the replicated genomic DNA. This contrasts with previous data that shows the non-amplified DHFR gene replicates during the first quarter of S phase. Therefore, we conclude that gene amplification alters the replication timing of the DHFR gene in CHOC 400 cells.     

Gene-specific and strand-specific DNA repair in the G1 and G2 phases of the cell cycle.

We have analyzed the fine structure of DNA repair in Chinese hamster ovary (CHO) cells within the G1 and G2 phases of the cell cycle. Repair of inactive regions of the genome has been suggested to increase in the G2 phase of the cell cycle compared with other phases. However, detailed studies of DNA repair in the G2 phase of the cell cycle have been hampered by technical limitations. We have used a novel synchronization protocol (D. K. Orren, L. N. Petersen, and V. A. Bohr, Mol. Cell. Biol. 15:3722-3730, 1995) which permitted detailed studies of the fine structure of DNA repair in G2. CHO cells were synchronized and UV irradiated in G1 or early G2. The rate and extent of removal of cyclobutane pyrimidine dimers from an inactive region of the genome and from both strands of the actively transcribed dihydrofolate reductase (DHFR) gene were examined within each phase. The repair of the transcribed strand of the DHFR gene was efficient in both G1 and G2, with no major differences between the two cell cycle phases. Neither the nontranscribed strand of the DHFR gene nor an inactive region of the genome was repaired in G1 or G2. CHO cells irradiated early in G2 were more resistant to UV irradiation than cells irradiated in late G1. Since we found no major difference in repair rates in G1 and G2, we suggest that G2 resistance can be attributed to the increased time (G2 and G1) available for repair before cells commit to DNA synthesis.     

Chromatin assembly during DNA replication in somatic cells.

Newly replicated DNA is assembled into chromatin through two principle pathways. Firstly, parental nucleosomes segregate to replicated DNA, and are transferred directly to one of the two daughter strands during replication fork passage. Secondly, chromatin assembly factors mediate de-novo assembly of nucleosomes on replicating DNA using newly synthesized and acetylated histone proteins. In somatic cells, chromatin assembly factor 1 (CAF-1) appears to be a key player in assembling new nucleosomes during DNA replication. It provides a molecular connection between newly synthesized histones and components of the DNA replication machinery during the S phase of the cell division cycle.