Participation of the Human β-Globin Locus Control Region in Initiation of DNA Replication

Abstract

The human beta-globin locus control region (LCR) controls the transcription, chromatin structure, and replication timing of the entire locus. DNA replication was found to initiate in a transcription-independent manner within a region located 50 kilobases downstream of the LCR in human, mouse, and chicken cells containing the entire human beta-globin locus. However, DNA replication did not initiate within a deletion mutant locus lacking the sequences that encompass the LCR. This mutant locus replicated in the 3' to 5' direction. Thus, interactions between distantly separated sequences can be required for replication initiation, and factors mediating this interaction appear to be conserved in evolution.

Overreplication of short DNA regions during S phase in human cells

“…This is consistent with the results for TOP1 and ␤-globin ORIs, where overabundant molecules derive precisely from regions where Orc2 binds and the polarity of nascent strand switches in these two ORIs, respectively (Figs. 2, 5;Aladjem et al 1995;Keller et al 2002). Alternatively, the generation of short molecules could take place somewhere in the vicinity of the replication initiation sites and might not participate in the generation of the mature daughter strands.…”

“…5A). In addition, an ORI that is active even in cells where the gene is not expressed has been located previously in a 1200-bp region encompassing the promoter and first exon of the gene (Kitsberg et al 1993;Aladjem et al 1995). We analyzed by Q-PCR the six regions of the HBB gene shown in the diagram of Figure 5B using total unfractionated DNA as a template and the cloned regions for normalization.…”

SV40 T antigen interacts with Nbs1 to disrupt DNA replication control

“…It appears likely that the rereplication suppression function of Nbs1 is inhibited following SV40 T/ Nbs1 complex formation. Yet, because T was required to maximize endoreduplication in NBS-deficient cells, A BAC clone containing a large DNA segment (clone RP11-645I8) of the ␤-globin locus, which includes a DNA replication initiation site (Kitsberg et al 1993;Aladjem et al 1995), was used as a FISH probe. a Interphase nuclei from primary IMR90 cells, NBS-deficient GM07166 cells (NBS), and wild-type (wt) Nbs1-reconstituted GM07166 cells [NBS −/− (Nbslwt)] were analyzed by FISH using this probe.…”

“…To more directly test the Nbs1 replication control hypothesis, we performed Fluorescence In Situ Hybridization (FISH) analysis on interphase cells using a DIG-labeled probe for sequences at a specific site near the ␤-globin locus where DNA replication is initiated (Kitsberg et al 1993;Aladjem et al 1995;Avni et al 2003). FITCconjugated secondary antibody against DIG was used to detect replication initiation foci at this site ( Fig.…”

“…Fixed cells were hybridized with a BAC probe corresponding to a segment covering the ␤-globin locus (clone RP11-645I8), and the hybridization images appear as green spots. This cloned genomic segment contains a putative replication origin (Kitsberg et al 1993;Aladjem et al 1995). Chromosomal DNA was stained with DAPI (blue).…”