Transitions in replication timing in a 340 kb region of human chromosomal R‐Band 1p36.1

Abstract

DNA replication is initiated within a few chromosomal bands as normal human fibroblasts enter the S phase. In the present study, we determined the timing of replication of sequences along a 340 kb region in one of these bands, 1p36.13, an R band on chromosome 1. Within this region, we identified a segment of DNA (approximately 140 kb) that is replicated in the first hour of the S phase and is flanked by segments replicated 1-2 h later. Using a quantitative PCR-based assay to measure sequence abundance in size-fractionated (900-1,700 nt) nascent DNA, we mapped two functional origins of replication separated by 54 kb and firing 1 h apart. One origin was found to be functional during the first hour of S and was located within a CpG island associated with a predicted gene of unknown function (Genscan NT_004610.2). The second origin was activated in the second hour of S and was mapped to a CpG island near the promoter of the aldehyde dehydrogenase 4A1 (ALDH4A1) gene. At the opposite end of the early replicating segment, a more gradual change in replication timing was observed within the span of approximately 100 kb. These data suggest that DNA replication in adjacent segments of band 1p36.13 is organized differently, perhaps in terms of replicon number and length, or rate of fork progression. In the transition areas that mark the boundaries between different temporal domains, the replication forks initiated in the early replicated region are likely to pause or delay progression before replication of the 340 kb contig is completed.

A novel role for fanconi anemia (FA) pathway effector protein FANCD2 in cell cycle progression of untransformed primary human cells

“…As expected, in control (siCon) HDF, we detected BrdU incorporation into the 18K and locus 15-19 hr after serum stimulation, time points corresponding to early S-phase. 31 Also as expected, the 74.5K, 31 FMR2, 32 and β-globin 33 loci replicated relatively late in S-phase (Fig. 6B).…”

“…After phenol/chloroform extraction and ethanol precipitation, immunoprecipitated DNA was resuspended in 50 ml of distilled water and analyzed by PCR. Sequences of the PCR primers we used to amplify the betaglobin, 26 18K, 74.5K, 31 and FMR2 32 loci are 5'-TCC TGA GGA GAA GTC TGC CGT TAC-3' (forward)/5'-CAG TGC AGC TCA CTC AGT GTG G-3' (reverse), 5'-CGT TTC CCA TGA TAA TAC AG 3' (forward)/5'-GAG AAA TGC ATA GGA AAA CC-3' (reverse), 5'-GTT TTC ACA CCT ACC ACG TT-3' (forward)/5'-TGT GGC TAC TTA ACG TCT CA-3' (reverse), and 5'-TGG GTG TTT TAT GGA GAA AC 3' (forward)/5'-GTC CTA GGC ATG TAA TTT GC-3' (reverse). The amplified DNA products were separated on 1.5% agarose gels and visualized under a UV transilluminator.…”

Isochores and replication time zones: a perfect match

“…A pause during replication has already been described for chromosome band boundaries in the human MHC (Tenzen et al, 1997) and on human chromosome 1p36.13 (Brylawski et al, 2004), and for an isochore boundary in the NF1 gene region of both, human and mouse (Schmegner et al, 2005). Here, we present three further examples for isochore boundaries where the replication machinery comes to a halt.…”

Early replication and the apoptotic pathway

“…This order may not be precisely definable at the nucleotide level, since in some regions origins of replication that are closely spaced may not be active in every cell cycle (DePamphilis, 1993). When examining a population of cells, however, one can clearly define a temporal order of replication with replication timing markers placed less than 25 kb apart (Tenzen et al, 1997;Brylawski et al, 2004). It has also long been known that there is a general correlation between a gene's replication time and whether it is actively transcribed (Holmquist, 1987).…”