In vivo protein-DNA interactions at human DNA replication origin.

Abstract

Protein-DNA interactions were studied in vivo at the region containing a human DNA replication origin, located at the 3' end of the lamin B2 gene and partially overlapping the promoter of another gene, located downstream. DNase I treatment of nuclei isolated from both exponentially growing and nonproliferating HL-60 cells showed that this region has an altered, highly accessible, chromatin structure. High-resolution analysis of protein-DNA interactions in a 600-bp area encompassing the origin was carried out by the in vivo footprinting technique based on the ligation-mediated polymerase chain reaction. In growing HL-60 cells, footprints at sequences homologous to binding sites for known transcription factors (members of the basichelix-loop-helix family, nuclear respiratory factor 1, transcription factor Spl, and upstream binding factor) were detected in the region corresponding to the promoter of the downstream gene. Upon conversion of cells to a nonproliferative state, a reduction in the intensity of these footprints was observed that paralleled the diminished transcriptional activity of the genomic area. In addition to these protections, in close correspondence to the replication initiation site, a prominent footprint was detected that extended over 70 nucleotides on one strand only. This footprint was absent from nonproliferating HL-60 cells, indicating that this specific protein-DNA interaction might be involved in the process of origin activation. ceding another still uncharacterized gene, provisionally named ppvl (13-15). The same origin, originally identified in HL-60 cells, was found to be active also in several other human cell types, including activated peripheral lymphocytes (S. Kumar, M.G., G.B., S.R., and A.F., unpublished results). A map of the 13.7-kb genomic fragment containing the replication origin is shown in Fig. 1A.The finding that the lamin B2 ori is located in a highly transcribed region is not surprising, since numerous reports have implicated transcriptional regulatory elements in the control of DNA replication (16)(17)(18)(19). Moreover, the domain is replicated at the very beginning of the S phase (12), consistent with the well-documented correlation between transcriptionally active genomic regions and early replication.We have now addressed the study of the specific protein-DNA interactions at the lamin B2 ori. To this purpose, we have exploited the in vivo footprinting technique based on the ligation-mediated polymerase chain reaction (LMPCR) (20).In the past few years we have extensively utilized this technique (21), which, in comparison with in vitro binding studies, is particularly suitable for the detection of the protein-DNA interactions actually occurring in the nucleus, since it is sensitive to the accessibility of sites to protein factors and possible epigenetic modification of DNA such as methylation. Furthermore, we have taken advantage of the property of HL-60 myeloid cells to undergo terminal differentiation upon chemical stimulation (22), a process which is accom...

Multi-step Loading of Human Minichromosome Maintenance Proteins in Live Human Cells

“…MCM proteins were shown to bind early firing origins earlier in G 1 phase compared with late-firing origins (66). Using FLIP, we observed the accumulation of MCMs at late-replicating heterochromatic regions from early G 1 phase in human cells (3 h post-mitotic release) and throughout G 1 phase, in line with previous studies on fixed cells (67). This suggests that if such a differential timing of MCM association with late replicating regions takes place in human cells, then it is confined to mitotic exit and very early G 1 phase.…”

High-Resolution Mapping of the Origin of DNA Replication in the Hamster Dihydrofolate Reductase Gene Domain by Competitive PCR

“…High-resolution mapping is required to study the protein-DNA interactions underlying the origin activation event by using in vivo footprinting methods (15).…”

Modular Structure of the Human Lamin B2 Replicator

“…Ligation-mediated PCR was performed with primer sets D and HIV specific for the endogenous and ectopic lamin B2 origins, respectively. Sequences and amplification conditions for both primers sets have been previously described (16,18). Two micrograms of DMS-treated genomic DNA was annealed to 0.5 pmol of primer HIV A1 (5Ј-TATTGAGGCTTAAGCAGTGGGTTCC-3Ј) specific for the ectopic origin by denaturation at 95°C for 5 min and incubation at 60°C for 30 min in 10 l of 1ϫ ThermoPol buffer [10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 20 mM Tris-HCl (pH 8.8), 2 mM MgSO 4 , 0.1% Triton X-100] combined with 6 mM MgSO 4 .…”