Gerald B. Price

Inverted repeats, stem‐loops, and cruciforms: Significance for initiation of DNA replication

Inverted repeats occur nonrandomly in the DNA of most organisms. Stem‐loops and cruciforms can form from inverted repeats. Such structures have been detected in pro‐ and eukaryotes. They may affect the supercoiling degree of the DNA, the positioning of nucleosomes, the formation of other secondary structures of DNA, or directly interact with proteins. Inverted repeats, stem‐loops, and cruciforms are present at the replication origins of phage, plasmids, mitochondria, eukaryotic viruses, and mammalian cells. Experiments with anti‐cruciform antibodies suggest that formation and stabilization of cruciforms at particular mammalian origins may be associated with initiation of DNA replication. Many proteins have been shown to interact with cruciforms, recognizing features like DNA crossovers, four‐way junctions, and curved/bent DNA of specific angles. A human cruciform binding protein (CBP) displays a novel type of interaction with cruciforms and may be linked to initiation of DNA replication.

Concurrent Replication and Methylation at Mammalian Origins of Replication

ABSTRACT Observations made with Escherichia coli have suggested that a lag between replication and methylation regulates initiation of replication. To address the question of whether a similar mechanism operates in mammalian cells, we have determined the temporal relationship between initiation of replication and methylation in mammalian cells both at a comprehensive level and at specific sites. First, newly synthesized DNA containing origins of replication was isolated from primate-transformed and primary cell lines (HeLa cells, primary human fibroblasts, African green monkey kidney fibroblasts [CV-1], and primary African green monkey kidney cells) by the nascent-strand extrusion method followed by sucrose gradient sedimentation. By a modified nearest-neighbor analysis, the levels of cytosine methylation residing in all four possible dinucleotide sequences of both nascent and genomic DNAs were determined. The levels of cytosine methylation observed in the nascent and genomic DNAs were equivalent, suggesting that DNA replication and methylation are concomitant events. Okazaki fragments were also demonstrated to be methylated, suggesting that the rapid kinetics of methylation is a feature of both the leading and the lagging strands of nascent DNA. However, in contrast to previous observations, neither nascent nor genomic DNA contained detectable levels of methylated cytosines at dinucleotide contexts other than CpG (i.e., CpA, CpC, and CpT are not methylated). The nearest-neighbor analysis also shows that cancer cell lines are hypermethylated in both nascent and genomic DNAs relative to the primary cell lines. The extent of methylation in nascent and genomic DNAs at specific sites was determined as well by bisulfite mapping of CpG sites at the lamin B2, c-myc, and β-globin origins of replication. The methylation patterns of genomic and nascent clones are the same, confirming the hypothesis that methylation occurs concurrently with replication. Interestingly, the c-mycorigin was found to be unmethylated in all clones tested. These results show that, like genes, different origins of replication exhibit different patterns of methylation. In summary, our results demonstrate tight coordination of DNA methylation and replication, which is consistent with recent observations showing that DNA methyltransferase is associated with proliferating cell nuclear antigen in the replication fork.

Major DNA replication initiation sites in the c-myc locus in human cells.

DNA replication initiation sites and initiation frequencies over 12.5 kb of the human c‐myc locus, including 4.6 kb of new 5′ sequence, were determined based on short nascent DNA abundance measured by competitive polymerase chain reaction using 21 primer sets. In previous measurements, no comparative quantitation of nascent strand abundance was performed, and distinction of major from minor initiation sites was not feasible. Two major initiation sites were identified in this study. One predominant site has been located at ∼0.5 kb upstream of exon 1 of the c‐myc gene, and a second new major site is located in exon 2. The site in exon 2 has not been previously identified. In addition, there are other sites that may act as less frequently used initiation sites, some of which may correspond to sites in previous reports. Furthermore, a comparison of the abundance of DNA replication intermediates over this same region of the c‐myc locus between HeLa and normal skin fibroblast (NSF) cells indicated that the relative distribution was very similar, but that nascent strand abundance in HeLa cells was approximately twice that in NSF relative to the abundance at the lamin B2 origin. This increased activity at initiation sites in the c‐myc locus may mainly be influenced by regulators at higher levels in transformed cells like HeLa. J. Cell. Biochem. 78:442–457, 2000.

The DNMT1 Target Recognition Domain Resides in the N Terminus

DNA-cytosine-5-methyltransferase 1 (DNMT1) is the enzyme believed to be responsible for maintaining the epigenetic information encoded by DNA methylation patterns. The target recognition domain of DNMT1, the domain responsible for recognizing hemimethylated CGs, is unknown. However, based on homology with bacterial cytosine DNA methyltransferases it has been postulated that the entire catalytic domain, including the target recognition domain, is localized to 500 amino acids at the C terminus of the protein. The N-terminal domain has been postulated to have a regulatory role, and it has been suggested that the mammalian DNMT1 is a fusion of a prokaryotic methyltransferase and a mammalian DNA-binding protein. Using a combination of in vitro translation of different DNMT1 deletion mutant peptides and a solid-state hemimethylated substrate, we show that the target recognition domain of DNMT1 resides in the N terminus (amino acids 122–417) in proximity to the proliferating cell nuclear antigen binding site. Hemimethylated CGs were not recognized specifically by the postulated catalytic domain. We have previously shown that the hemimethylated substrates utilized here act as DNMT1 antagonists and inhibit DNA replication. Our results now indicate that the DNMT1-PCNA interaction can be disrupted by substrate binding to the DNMT1 N terminus. These results point toward new directions in our understanding of the structure-function of DNMT1.

Ku antigen, an origin-specific binding protein that associates with replication proteins, is required for mammalian DNA replication.

Ors binding activity (OBA) represents a HeLa cell protein activity that binds in a sequence-specific manner to A3/4, a 36-bp mammalian replication origin sequence. OBAs DNA binding domain is identical to the 80-kDa subunit of Ku antigen. Ku antigen associates with mammalian origins of DNA replication in vivo, with maximum binding at the G1/S phase. Addition of an A3/4 double-stranded oligonucleotide inhibited in vitro DNA replication of p186, pors12, and pX24, plasmids containing the monkey replication origins of ors8, ors12, and the Chinese hamster DHFR oribeta, respectively. In contrast, in vitro SV40 DNA replication remained unaffected. The inhibitory effect of A3/4 oligonucleotide was fully reversed upon addition of affinity-purified Ku. Furthermore, depletion of Ku by inclusion of an antibody recognizing the Ku heterodimer, Ku70/Ku80, decreased mammalian replication to basal levels. By co-immunoprecipitation analyses, Ku was found to interact with DNA polymerases alpha, delta and epsilon, PCNA, topoisomerase II, RF-C, RP-A, DNA-PKcs, ORC-2, and Oct-1. These interactions were not inhibited by the presence of ethidium bromide in the immunoprecipitation reaction, suggesting DNA-independent protein associations. The data suggest an involvement of Ku in mammalian DNA replication as an origin-specific-binding protein with DNA helicase activity. Ku acts at the initiation step of replication and requires an A3/4-homologous sequence for origin binding. The physical association of Ku with replication proteins reveals a possible mechanism by which Ku is recruited to mammalian origins.

Identification of Initiation Sites for DNA Replication in the Human dnmt1 (DNA-methyltransferase) Locus

Vertebrates have developed multiple mechanisms to coordinate the replication of epigenetic and genetic information.Dnmt1 encodes the maintenance enzyme DNA-methyltransferase, which is responsible for propagating the DNA methylation pattern and the epigenetic information that it encodes during replication. Direct sequence analysis and bisulfite mapping of the 5′ region of DNA-methyltransferase 1 (dnmt1) have indicated the presence of many sequence elements associated with previously characterized origins of DNA replication. This study tests the hypothesis that thednmt1 region containing these elements is an origin of replication in human cells. First, we demonstrate that a vector containing this dnmt1 sequence is able to support autonomous replication when transfected into HeLa cells. Second, using a gel retardation assay, we show that it contains a site for binding of origin-rich sequences binding activity, a recently purified replication protein. Finally, using competitive polymerase chain reaction, we show that replication initiates in this region in vivo. Based on these lines of evidence, we propose that initiation sites for DNA replication are located between the first intron and exon 7 of the human dnmt1 locus.

Decreased origin usage and initiation of DNA replication in haploinsufficient HCT116 Ku80+/- cells.

One of the functions of the abundant heterodimeric nuclear protein, Ku (Ku70/Ku80), is its involvement in the initiation of DNA replication through its ability to bind to chromosomal replication origins in a sequence-specific and cell cycle dependent manner. Here, using HCT116 Ku80+/- cells, the effect of Ku80 deficiency on cell cycle progression and origin activation was examined. Western blot analyses revealed a 75% and 36% decrease in the nuclear expression of Ku80 and Ku70, respectively. This was concomitant with a 33% and 40% decrease in chromatin binding of both proteins, respectively. Cell cycle analysis of asynchronous and late G1 synchronized Ku80+/- cells revealed a prolonged G1 phase. Furthermore, these Ku-deficient cells had a 4.5-, 3.4- and 4.3-fold decrease in nascent strand DNA abundance at the lamin B2, β-globin and c-myc replication origins, respectively. Chromatin immunoprecipitation (ChIP) assays showed that the association of Ku80 with the lamin B2, β-globin and c-myc origins was decreased by 1.5-, 2.3- and 2.5-fold, respectively, whereas that of Ku70 was similarly decreased (by 2.1-, 1.5- and 1.7-fold, respectively) in Ku80+/- cells. The results indicate that a deficiency of Ku80 resulted in a prolonged G1 phase, as well as decreased Ku binding to and activation of origins of DNA replication.

The Human Cruciform-binding Protein, CBP, Is Involved in DNA Replication and Associates in Vivo with Mammalian Replication Origins

We previously identified and purified from human (HeLa) cells a 66-kDa cruciform-binding protein, CBP, with binding specificity for cruciform DNA regardless of its sequence. DNA cruciforms have been implicated in the regulation of initiation of DNA replication. CBP is a member of the 14-3-3 family of proteins, which are conserved regulatory molecules expressed in all eukaryotes. Here, the in vivo association of CBP/14-3-3 with mammalian origins of DNA replication was analyzed by studying its association with the monkey replication origins ors8 andors12, as assayed by a chromatin immunoprecipitation assay and quantitative PCR analysis. The association of the 14-3-3β, -ε, -γ, and -ζ isoforms with these origins was found to be ∼9-fold higher, compared with other portions of the genome, in logarithmically growing cells. In addition, the association of these isoforms with ors8 and ors12 was also analyzed as a function of the cell cycle. Higher binding of 14-3-3β, -ε, -γ, and -ζ isoforms with ors8 and ors12 was found at the G1/S border, by comparison with other stages of the cell cycle. The CBP/14-3-3 cruciform binding activity was also found to be maximal at the G1/S boundary. The involvement of 14-3-3 in mammalian DNA replication was analyzed by studying the effect of anti-14-3-3β, -ε, -γ, and -ζ antibodies in thein vitro replication of p186, a plasmid containing the minimal replication origin of ors8. Anti-14-3-3ε, -γ, and -ζ antibodies alone or in combination inhibited p186 replication by ∼50–80%, while anti-14-3-3β antibodies had a lesser effect (∼25–50%). All of the antibodies tested were also able to interfere with CBP binding to cruciform DNA. The results indicate that CBP/14-3-3 is an origin-binding protein, acting at the initiation step of DNA replication by binding to cruciform-containing molecules, and dissociates after origin firing.

Anti-cruciform DNA affinity purification of active mammalian origins of replication

A novel approach that employs anti-cruciform DNA monoclonal antibodies was used to isolate segments of cruciform-containing DNA from genomic DNA, in an effort to obtain fragments containing active origins of replication. High molecular weight DNA (greater than 50 kb) was extracted from log phase CV-1 cells and 6 micrograms incubated with approximately 2.5 micrograms of a monoclonal antibody, 2D3, specific for cruciform-containing DNA. The 2D3-bound DNA was digested with EcoRI and antibody-bound fragments were recovered using rabbit anti-mouse immunobeads. The beads were washed free of nonspecifically bound DNA and the 2D3-bound DNA was eluted with 2% sodium dodecyl sulphate (SDS). The yield of DNA recovered by 2D3 was 2000-fold less than the initial amount and was 17-20-fold more than that recovered nonspecifically using the control mAb, P3. The 2D3-bound DNA ranged from 0.15- greater than 23 kb with a major peak at approximately 12 kb. Specific enrichment of origin-containing DNA by 2D3 over P3 was suggested by a 10-100-fold greater recovery of a 9 kb fragment hybridizable to a low-copy monkey autonomously replicating sequence, ors 8. 20 ng of affinity-purified DNA was cloned into lambda Zap II and excised into Bluescript phagemids in vivo. Of nine randomly-selected clones between 0.15 and 3.2 kb, four were able to replicate autonomously when transfected into HeLa cells. Two of the nine clones contained sequences hybridizable to both monkey alpha-satellite and human Alu DNA, and two others to Alu alone. The present work provides further evidence for the involvement of cruciforms at active mammalian origins of DNA replication.

Slipped (CTG)·(CAG) Repeats of the Myotonic Dystrophy Locus: Surface Probing with Anti-DNA Antibodies

At least 15 human diseases have been associated with the length-dependent expansion of gene-specific (CTG).(CAG) repeats, including myotonic dystrophy (DM1) and spinocerebellar ataxia type 1 (SCA1). Repeat expansion is likely to involve unusual DNA structures. We have structurally characterized such DNA, with (CTG)(n).(CAG)(n) repeats of varying length (n=17-79), by high-resolution gel electrophoresis, and have probed their surfaces with anti-DNA antibodies of known specificities. We prepared homoduplex S-DNAs, which are (CTG)x.(CAG)y where x=y, and heteroduplex SI-DNAs, which are hybrids where x>y or x<y. S-DNAs formed many different species of slipped isomers, as indicated by its multiple electrophoretic species. In contrast, SI-DNAs formed distinct structures, as indicated by the limited electrophoretic species for all possible repeat length pairings. Sister SI-DNAs with an excess of CAG repeats always migrated slower than their sister SI-DNAs with an excess of CTG repeats. Strikingly, both the propensity to form slipped structures and the pattern of S-DNAs, but not SI-DNAs, varied for similar lengths of CTG/CAG repeats between the DM1 and SCA1 loci, highlighting a role for flanking cis-elements in S-DNA but not SI-DNA formation. Slipped structures bound structure and nucleotide-specific anti-DNA antibodies. Binding of anti-B-DNA antibodies was reduced for both S-DNAs and SI-DNAs relative to their linear forms. SI-DNAs bound anti-Z-DNA antibodies, while both S and SI-DNAs bound anti-cruciform antibodies, revealing shared characteristics between the corresponding DNA structures and slipped DNAs. Such features of the repeats may be recognized by cellular proteins known to bind such structures.