The coordinate replication of the human beta-globin gene domain reflects its transcriptional activity and nuclease hypersensitivity.

Abstract

The temporal order of replication of DNA sequences in the chromosomal domain containing the human j3-globin gene cluster and its flanking sequences (140 kilobases) was measured and compared in two different human cell lines. In human erythroleukemia (K562) cells, in which embryonic and fetal globin genes are transcribed, all of the sequences we examined from the P-globin domain replicated early during S phase, while in HeLa cells, in which globin genes are transcriptionally silent, these sequences replicated late during S. Potential sites of initiation of DNA replication within this domain were identified. The 3-globin gene domain was also found to differ with respect to the nuclease sensitivity of the chromatin in these two cell lines. In K562 cells, hypersensitive sites for endogenous nucleases and DNase I were present in the chromatin near the earliest-replicating segments in the 13-globin domain.In viruses and procaryotes, DNA replication initiates at well-defined sequences (origins of replication) and proceeds bidirectionally. (IgCH) in mouse cell lines is also consistent with replication proceeding from a presumptive origin downstream of the gene cluster when the locus is inactive (5). However, when one or more genes in the IgCH cluster are transcribed, the entire cluster replicates at the same time at the beginning of S with no apparent temporal directionality (5). Thus, a change in the timing and possibly a change in the origin and direction of replication is associated with the change in the transcriptional activity within this cluster.To gain more information about the relationship between gene expression and the timing of DNA replication in mammalian cells, we have studied the human ,B-globin gene cluster. This gene cluster has been well characterized at the molecular level and consists of an embryonic (E) gene expressed in the early embryo, two fetal genes (Gy, AY) expressed in the fetal liver, two adult genes (8, I) expressed in adult bone marrow, and a pseudogene (i4. These genes undergo a switch in their expression during erythroid cell development. These genes are clustered in a 50-kilobase (kb) region on chromosome 11 and occur in the order 5'-Gyy8Ab-3' (11). Centrifugal elutriation was used to fractionate 5-bromodeoxyuridine (BUdR)-labeled human cells into various intervals of S phase, followed by detailed hybridization analysis of bromouracil (BU)-labeled, newly replicated DNA with DNA probes spanning the length of the human ,B-globin gene locus. This fractionation technique has a number of distinct advantages over other assays; it is more rapid, a large number of cells can be fractionated, and most importantly, it obviates the need for the toxic chemicals that are used in cell synchronization. In this report, we show that the timing of replication during the S phase of a contiguous stretch of chromosomal DNA (140 kb) containing the human 4958

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

“…In eukaryotes, genes that are transcribed early in S phase also replicate early (41) and chromosomal segments that are heterochromatic tend to replicate late (43). Orc proteins, which have been implicated in initiation of chromosome replication, also contribute to silencing of transcription of yeast mating type cassettes (42).…”

Human Timeless and Tipin stabilize replication forks and facilitate sister-chromatid cohesion

“…However, we were not able to detect significant Timeless association at regions flanking the MCM4 origin, possibly because of the fast movement of replication forks. We also monitored a replication origin located at the b-globin gene locus (HBB), which is known to fire late in S phase in HeLa cells, but early in S phase in erythroleukemia cells (Dhar et al, 1988). Unexpectedly, Timeless was detected at the b-globin origin region during early S phase (0 and 1 hour), and this association disappeared by 2 hours after the release (Fig.…”

Replication profile of Saccharomyces cerevisiae chromosome VI

“…Genes highly expressed in a given cell type tend to be early replicating, while the same genes in a transcriptionally quiescent state in other cell types tend to replicate later in S phase (e.g. Dhar et al 1988;Epner et al 1988). The causal relationship between transcriptional activation and replication time is not understood, however.…”